Cumitech 16A: Laboratory Diagnosis of the Mycobacterioses

OCTOBER

1994

Laboratory Diagnosis of the Mycobacterioses PATRICIA L. CERNOCH, RUSSEL K. ENNS, MICHAEL A. SAUBOLLE, and RICHARD J. WALLACE, JR. COORDINATING

EDITOR

ALICE S. WEISSFELD

AMERICAN

SOCIETY

FOR MICROBIOLOGY

Cumitech 1A l Blood Cultures II l June 1982 Cumitech 2A l Laboratory Diagnosis of Urinary Tract Infections l March 1987 Cumitech 3A l Quality Control and Quality Assurance Practices in Clinical Microbiology l May 1990 Cumitech 4A l Laboratory Diagnosis of Gonorrhea l April 1993 Cumitech 5A l Practical Anaerobic Bacteriology l December 1991 Cumitech 6A l New Developments in Antimicrobial Agent Susceptibility Testing: a Practical Guide l February 1991 Cumitech 7A l Laboratory Diagnosis of Lower Respiratory Tract Infections l September 1987 Cumitech 8 l Detection of Microbial Antigens by Counterimmunoelectrophoresis l December 1978 Cumitech 9 l Collection and Processing of Bacteriological Specimens l August 1979 Cumitech 10 l Laboratory Diagnosis of Upper Respiratory Tract Infections l December 1979 Cumitech 11 l Practical Methods for Culture and Identification of Fungi in the Clinical Microbiology Laboratory l August 1980 Cumitech 12A l Laboratory Diagnosis of Bacteria1 Diarrhea l April 1992 Cumitech 13A l Laboratory Diagnosis of Ocular Infections l September 1994 Cumitech 14A l Laboratory Diagnosis of Central Nervous System Infections l March 1993 Cumitech 15A l Laboratory Diagnosis of Viral Infections l August 1994 Cumitech 16A l Laboratory Diagnosis of the Mycobacterioses l October 1994 Cumitech 17A l Laboratory Diagnosis of Female Genital Tract Infections l June 1993 Cumitech 18 l Laboratory Diagnosis of Hepatitis Viruses l January 1984 Cumitech 19 l Laboratory Diagnosis of Chlamydial and Mycoplasmal Infections l August 1984 Cumitech 20 l Therapeutic Drug Monitoring: Antimicrobial Agents l October 1984 Cumitech 21 l Laboratory Diagnosis of Viral Respiratory Disease l March 1986 Cumitech 22 l Immunoserology of Staphylococcal Disease l August 1987 Cumitech 23 l Infections of the Skin and Subcutaneous Tissues l June 1988 Cumitech 24 l Rapid Detection of Viruses by Immunofluorescence l August 1988 Cumitech 2.5 l Current Concepts and Approaches to Antimicrobial Agent Susceptibility Testing l December 1988 Cumitech 26 l Laboratory Diagnosis of Viral Infections Producing Enteritis l September 1989

Cumitechsshould becited as follows, e.g.: Cernoch, Cumitech 16A, Laboratory diagnosis of the mycobacterioses. Microbiology,

Editorial Janet Handler, Stephen Young

Washington,

Board Thomas

P. L., R. K. Enns, Coordinating

M. A. Saubolle, and ed., A. S. Weissfeld.

R. J. Wallace, American

Jr. 1994. Society for

D.C.

for

ASM J. Inzana,

Cumitechs: Brenda

Steven McCurdy,

C.

Specter,

Frederick

Charman; S. Nolte,

Mary John

J. R

A. Smtth,

Gtlchrtst, Alice

Curt S

Gleaves,

Wetssfeld,

The purpose of the Cumitech series is to provrde consensus recommendations by the authors as to appropriate state-of-the-art operating procedures for clinical microbiology laboratories which may lack the facilities for fully evaluating routine or new methods. The procedures given are not proposed as “standard” methods.

Copyrtgnt

0 1994 Amertcan Soctety for MIcrobIology 1325 Massachusetts Ave , N W Washington DC 20005

and

LABORATORY DIAGNOSIS OF THE MYCOBACTERIOSES PATRICIA RUSSEL

L. CERNOCH, K. ENNS,

The Methodist

Microprobe

MICHAEL

A. SAUBOLLE,

RICHARD

J. WALLACE,

Hospital,

Corporation, Good

Samaritan

JR., The University

Houston,

Bothell, Regional

Washington Medical

of Texas Health COORDINATING

ALICE S. WEISSFELD, Microbiology Specialists Incorporated Baylor College of Medicine, Houston, Texas 77054

SYNOPSIS

The past decade has seen the resurgenceof Mycobacterium tuberculosis, the discoveryof new, pathogenicspeciesof mycobacteria,and the introduction of new technological advancesfor the rapid detection and identification of acid-fastorganisms.This Cumitech examinesthesenew developments in the context of a rational, costeffective approachto the diagnosisand treatment of theseorganisms.Safety issuesand quality assuranceparametersare alsoaddressed. As with the first edition of this Cumitech on the laboratory diagnosisof the mycobacterioses, published in 1983 (IlO), this update is designedto provide laboratory personnel,includingthosewith limited experiencein mycobacteriology,the information they need to accurately and confidently work with mycobacteriain the clinical laboratory. Actual techniquesare given in several publications (77, 98). Since the publication of the first edition of this Cumitech 11 years ago, there have been dramatic technical advancesthat include a radiometric detection methodology, the use of species-specificDNA probes and high-performanceliquid chromatographic(HPLC) methods for identification, andnewdrugsand susceptibility test methods for some of the nontuberculous mycobacteria(NTM). This Cumitech includesinformation regardingboth new and conventional techniquesapplicableto the recovery and identification of specificorganismsand to the performanceof susceptibilitytesting where indicated. Several termshave been usedto describemycobacteriathat do not belong to the Mycobacterium tuberculosis complex. The term nontuberculows mycobacteria is preferable to the terms anonymous,atypical, mycobacteriaother than tuberculosis,and opportunistically pathogenic mycobacteria and will be used throughout this Cumitech.

Texas 77030 98021 Center, Center

Phoenix,

Arizona

85006

at Tyler, Tyler, Texas 75710

EDITOR and Department

of Microbiology

CLINICAL

and Immunology,

SYNDROMES

Tuberculosisand diseases due to the NTM have undergonea striking changein the past 10years, largely as a consequenceof the human immunodeficiency virus (HIV) epidemic. A continuous annualdeclinein the number of casesof tuberculosisin the United Statesduring the past20 years was interrupted in 1985,when the expected decline in numbers of casesfailed to occur (38). Over the next 2 years,the numberof casesactually increased.Cross-referencingthe registry of patients with AIDS with the registry of patientswith tuberculosisshowed that approximately 5% of patients with AIDS also developedtuberculosis. This method underestimatesthe associationof HIV diseaseand tuberculosis,as most casesof tuberculosisoccur in individuals who are still asymptomaticfrom their HIV diseaseand have CD4 lymphocyte counts in excessof 100cellsper l.~l(57). In studiesin areasof the country where HIV diseaseand tuberculosisare common, between 20 and 40% of patients diagnosedwith tuberculosis are HIV positive (40). The HIV epidemic may not be the only reason for the increasein casesof tuberculosis;i.e., incidencein somegroups,suchasminorities, immigrants,and the homeless,has shownlittle or no declineover the last 10 years, while incidence in the general population of the United Stateshasbeen declining (78). The Centers for DiseaseControl and Prevention (CDC) and the American Thoracic Society (ATS) currently recommenda shortened tuberculosistreatment of 6 months in the nonAIDS patient (isoniazid and rifampin for 6 months;pyrazinamidefor the first 2 months)in an attempt to increasethe number of patients who completea satisfactorycourseof therapy (3). An additional challengehas been the emergenceof multidrug-resistanttuberculosis,which occursprimarily in New York and Florida (38). A variety of diseases, including pulmonary disease,skin and soft tissueinfections, lymphadeni-

2

CERNOCH

ET AL.

tis, and disseminated disease, are produced by the NTM (91, 123, 126, 137). Pulmonary disease is probably the best-known and first-recognized disease due to these organisms. 1M. avium complex (MAC), which may includeboth M. avium andM. intracellulare, is the mostcommonpathogenin this group, followed by M. kansasii, M. abscessus (formerly M. chelonae subsp.abscessus), M. fortuitum, M. simiae, M. xenopi, and, rarely, other organisms. Most patientswith pulmonary diseaseare over 50 years of age and, except for chronic lung disease due to smoking,have few underlying diseases. As many as 50% of patientswith NTM lung disease have no specificrisk factor for their disease.Skin and soft tissuediseases due to the NTM are being recognizedwith increasingfrequency. Although fewer than 200 casesof leprosy are diagnosedin the United States each year, diseasedue to M. Zeprae remainsa major causeof morbidity and mortality worldwide. Rifampin has produced a dramatic improvementin therapy of leprosy, but its cost haslimited its availability in Third World countries.M. marinum causesposttraumaticskin andsoft tissueinfectionsof the extremitiesfollowing exposureto contaminatedsalt or fresh water, primarily in the southeasternUnited States(137). M. fortuitum, M. abscessus, and M. chelonae (formerlyM. chelonae subsp.chelonae) causea variety of cutaneous infections (124, 126), and these organismsalong with M. marinum are the most commoncausesof primary cutaneousmycobacterial diseasein this country. Other occasionalcutaneouspathogensinclude MAC, M. terrae complex (97), M. kansasii, and M. smegrnatis (122, 137).A newly recognizeddisseminatedcutaneous infection in patients receiving organ transplants and thosewith AIDS is due to M. haemophilum (33, 64). NTM lymphadenitisis seenprimarily in childrenbetween1 and 5 years of age (137). The diseaseis localized to the cervical lymph nodes and is usuallyreadily treated by surgicalexcision. Currently, approximately 80% of these casesare due to MAC (73, 92). DisseminatedMAC infection occursin asmany as40% of AIDS patientswith low (~50 cellsper ~1) CD4 lymphocyte counts (89). This is a relatively new disease,asno more than 20 caseswere recognizedbefore the HIV epidemic(51). AIDS patientswith disseminated diseaseusuallyhaveno evident skin or lung disease;instead, the organismsare concentratedin the gastrointestinaltract, bonemarrow,liver, and spleen(54). Although less common,a poorly growing speciesknown as M. genavense causesa similar diseasein the same setting as MAC (27). Diagnosisof disseminated infection dueto either organismcurrently involves cultures of blood and sometimesbone marrow and stool (54, 59), a methodologythat wasrarely if ever usedin the past.

CUMITECH

16~4

CLASSIFICATION

Classic laboratory descriptions of the NTM have included organization of the various species into four groupson the basisof growth rates and pigmentation(137).Organismsthat took lessthan 7 daysto grow isolatedcolonieswere calledrapid growers,while thosethat took more than 7 days were called slowgrowers.In this system,devised by Ernst Runyon, the developmentof pigmentation was evaluated both with and without exposure to light. Slow-growing photochromogens (coloniespigmentedonly after exposureto light, e.g., M. kansasii and M. marinum) were called group I; slow-growingscotochromogens (colonies pigmentedin the dark, e.g., M. scrofilaceum and M. gordonae) were called group II; slow-growing nonphotochromogens(e.g., M. avium complex) were called group III; and rapidly growing mycobacteria (primarily M. fortuitum, M. chelonae, and M. abscessus) were called group IV. This classification systemwasuseful when only six or seven speciesof NTM were encounteredin the routine laboratory. Currently, there are more than 20 recognizedpathogens,eachone unique, and each with a unique clinical significance.Becauseof the large number of species,many people find it difficult to rememberwhich speciesbelongin each Runyon group. Moreover, somespecieswithin the Runyon systemare not easilyclassified.For example, most of the M. avium isolatesfrom AIDS patients are pigmentedand would be incorrectly listed in group II. Likewise,M. szulgai may show scotochromogenicityat 35°Candphotochromogenicity at 25°C. For thesereasons,this classificationsystemof the nontuberculousspecieshas generally been replacedby a newersystembasedon the typesof diseasethe speciesproduce,suchaspulmonary or cutaneousdisease(Table 1). This approachwas taken by the American Thoracic Society (ATS) in their recent statementon the diagnosisand treatment of the NTM (123). This disease-oriented systemof classificationmay offer more advantages to the clinician and laboratory personnel,as they think of potential pathogens according to the source of the clinical sample (sputum, wound, blood, etc.) rather than just morphologicfeatures. For example,blood isolatesof NTM have a very limited differential with MAC being responsible for mostisolates.Likewise,cutaneousdisease brings to mind a number of different pathogens,including M. marinum and M. haemophilum, which are more commonthan other speciesin this source. LEVELS

OF SERVICE

As the number of primary mycobacteriallaboratories has declined, many general bacteriology laboratorieshave taken over the responsibilityof handling mycobacterialcultures:becausethe vol-

CUMITECH

16A

DIAGNOSIS TABLE

Clinical disease

Pulmonary

Lymphadenitis

Cutaneous

OF THE MYCOBACTERIOSES

1. Classification of the NTM recovered from humans”

Common etiologic species

MAC

Growth rate

M. kansasii

Slow (>7 days) Slow

M. abscessus

MAC

Morphologic feature@

Less frequent etiologic species M. xenopi M. simiae

Rapid (~7 days)

Usually not pigmented Photochromogen; often large and beaded on AFB smear Not pigmented

M. M. M. M. M.

szulgai malmoense fortuitum smegmatis chelonae

M. scrofilaceum

Slow Slow

Usually not pigmented Scotochromogen

M. M. M. M. M.

fort&urn chelonae abscessus kansasii haemophilum

M. marinum

Slow

Photochromogen; requires low temp (28-30°C) for isolation Not pigmented Not pigmented

MAC

M. fortuitum M. fortuitum

3

third

Rapid Rapid

M. kansasii

biovariant complex

Disseminated

M. chelonae M. abscessus M. ulcerans

Rapid Rapid Slow

M. haemophilum

Slow

MAC

Slow

M. kansasii M. chelonae M. haemophilum

Slow Rapid Slow

Not pigmented Not pigmented Usually scotochromogen; requires low temp for isolation Not pigmented

M. nonchromogenicum M. peregrinum M. smegmatis

Isolates from patients with AIDS; usually pigmented (80%) Photochromogen Not pigmented Not pigmented; requires hemin; often needs low temp and CO, to grow

M. fortuitum

M. abscessus M. xenopi M. gordonae

M. genavense

a Modified from reference 123, b Photochromogen, isolate is buff in the dark but turns yellow with brief exposure to light; scotochromogen, isolate is yellow-orange or orange even when grown in the dark.

umeof theseculturesand the numberof positives may be small,many laboratoriesdoing complete organismidentification and susceptibilitytesting would spendas much time on quality control as they would with actual patient isolates. Many low-volumelaboratorieseither do no mycobacterial work or perform acid-fast bacillus (APB) smearsand cultures, referring cultures demonstrating growth of mycobacteria to a reference laboratory for identification and susceptibility testing. The levels-of-serviceconcept was first introduced by the Public Health Service in 1967 and is supportedby severalgroups,including the CDC and ATS. In this system,laboratoriesselect the level that bestfits their clinical needs,personnel experience, laboratory facilities, and work

volume. The levels of serviceasproposedby the Collegeof American Pathologists(CAP) and recommendedby the ATS are shownin Table 2 (68). In general,the three ATS levelsof service(I, II, and III) are equivalentto extents 2, 3, and 4 used by CAP (84, 109, 110). It is currently estimated that the majority of clinical laboratoriesare ATS extent 2 and that only about 10% provide the full range of servicesdescribedin extent 3. The optimal numbers of acid-fast smearsand cultures neededto maintainproficiencywithin a laboratory are uncertain, but it has been suggestedthat laboratories that interpret APB smearsshould handle10to 15specimens per weekandthat those that perform cultures shouldprocess20 or more specimensper week.

4

CERNOCH ET AL. TABLE

CUMITECH

16A

2. Laboratory self-determined extents or levels of serviceQ

CAP extents of service for participation in mycobacterial interlaboratory comparison surveys

Extent 1 No mycobacterial procedures performed.

Extent 2 Acid-fast stain of exudates, effusions, and body fluids, etc., with inoculation and referral of cultures to reference laboratories for further identification.

Extent 3 Isolation of mycobacteria; identification of M. tuberculosis and preliminary identification of atypical forms as photochromogens, scotochromogens, nonphotochromogens, and rapid growers. Drug susceptibility testing may or may not be performed. Extent 4 Definitive identification of mycobacteria isolated to the extent required to establish correct clinical diagnosis and to aid in selection of safe and effective therapy. Drug susceptibility testing may or may not be performed.

ATS levels of service for mycobacterial

laboratories

Level I a. Collect adequate clinical specimens, including aerosol-induced sputa. b. Transport specimens to higher-level laboratory for isolation and identification. c. May prepare and examine smears for presumptive diagnosis and as means of monitoring progress of diagnosed patients on chemotherapy. Level II a. May perform all functions of level I laboratories and also process specimens as necessary for culture on standard agar-based and egg-based media. b. Identify M tuberculosis. c. May perform drug susceptibility studies of N: tuberculosis with primary antituberculous drugs. d. Retain mycobacterial cultures for reasonable time. Level III a. May perform all functions of laboratories at lower levels and also identify all Mycobacterium species from clinical specimens. b. Perform drug susceptibility studies of mycobacteria. c. Retain mycobacterial cultures for reasonable time. d. May conduct research and provide training.

a Taken from reference 110.

SAFETY Laboratoriesthat processspecimensfor detection, isolation, and identification of mycobacteria shouldensurethat safety measuresare appropriately implementedand enforced.Biosafety level 2 practicesshouldbe adheredto by facilities processingspecimensfor direct smearsand inoculation to isolation media. Such practices include wearing of gloves and other personalprotective equipment,appropriate hand washingafter specimen handling, avoidanceof generatingaerosols, and appropriate handling and discardingof needles, if their use is absolutely necessary,into puncture-resistantsharpscontainers. More rigorous biosafety level 3 guidelines should be followed by laboratories performing mycobacterial identifications and working with large inocula(6,21, 109).For working with mycobacterial cultures, a separate, dedicated room

maintainedat negative pressureshouldbe available.A laminar-flowbiologicalsafetycabinetwith a high-efficiencyparticulate air (HEPA) filtration systemand an adequateair draw acrossthe front mustbe presentin the room. This cabinetmay be a classIIA biological safety cabinet and should have the exhaustair vented directly to the outside or into an appropriate building exhaust system. Air in a cabinet may be recirculated within the laboratory if the cabinet is inspectedand certified at least annually. An autoclave should be available in an easily accessibleadjacent area; infectious waste should be decontaminatedbefore removal to disposal areas.High-speedcentrifugation systems,including cytocentrifuges, need to be equipped with bucket covers and domesto contain debris from possibletube breakage.To avoid splatter, electric incinerators are preferable to bunsen burners.

CUMITECH

16A

DIAGNOSIS

Straight wires,loops,or spadesshouldbe cleaned in a containerof sandand a 5% phenolsolutionor 95% alcohol solution to remove excessinoculum prior to incineration. Heat fixation of direct smearson a hot plate (65 to 80°C for 2 to 2.5 h) may not kill all of the mycobacteriallawn on the slides,and theseslides must be handled carefully (6, 98). Addition of sodiumhypochlorite (householdbleach) to mycobacterium-containingsputumto be usedfor preparation of smearswill kill mycobacteria. All work surfaces,includingtops of benchesand insidesof biological safety cabinets, should be cleanedwith appropriatedisinfectantsbefore and after work. To further decreaseaerosolizationand contamination,towels may be soakedwith disinfectants and placedin areaswhere work is done, or they may be usedto wipe the outsidesof tubes or containers.Appropriate disinfectant solutions may include sodiumhypochlorite at either a 1:200 or a l:l,OOO concentration prepared daily, 5% phenol, 5% formaldehyde,2% glutaraldehyde,or phenol-soapmixtures such as Staphene(98). All local regulations(e.g., state, county, or city ordinances)regarding useand discard of potentially hazardous chemicalsshould be followed when disinfectantsare handled. Gownsand glovesshouldbe worn when working with specimensor cultures; some workers further recommendwearingmasksand caps.Proceduresshouldbe performed in a manner minimizing aerosolproduction while maximizingprotection of personnelfrom infection by droplet nuclei.Personnelworking in the laboratory should undergo tuberculin skin tests at least annually to detect conversions;recent Occupational Safety and Health Administration regulations suggest biannualskintestsfor high-riskindividuals.Those with positiveskin testsshouldhave annualchestX rays. New converters should be referred to the Employee Health and Infection Control departmentsfor medicalandepidemiologicalevaluation. MAILING

The Code of Federal Regulations must be adheredto when specimens or cultures are packaged for mailing to reference laboratories (88). Watertight screw-captubes must be used. Raw specimens or pure isolatesshouldbe placedwithin a watertight metallic containerwith enoughabsorbent material to take up all the liquid in caseof breakageor leakage.One or more of thesespecimens can be packed into a secondwatertight container made of paperboard or wood. Specimensmay be mailed only after appropriate biohazardwarninglabelsare attachedto the outside of containers(6, 43, 84).

OF THE MYCOBACTERIOSES

SPECIMEN

5

COLLECTION

Methods of specimencollection and transport to the laboratory directly influence the detection and isolation of mycobacteria. All laboratories should provide information to maximize efficacy of collection techniquesand to expedite specimen transportation (Table 3). Many different types of specimensmay be submitted for isolationand/or detection of mycobacteria; the choice of specimenis commonly predicated on the patient’s clinical presentation.The majority of specimensare from the respiratory tract; tissue,body fluids, urine, and gastric aspirates may also be submitted. Specimensfrom AIDS patients may include blood and stool. To minimize culture overgrowth by commensalmicroorganisms,collection methodsshould bypass areas of colonization as much as possible.All specimensshouldbe refrigerated if processingwill be delayed. Potentially Respiratory

Contaminated

Specimens

tract

Specimenssubmitted for the diagnosisof pulmonary mycobacteriosisinclude expectorated as well as induced sputa, bronchial alveolar lavage fluids, gastric aspirates,and laryngeal swabbings. Transtracheal aspirationshave become uncommon, but invasivetechniquessuchas fine-needle biopsy or open-lungbiopsy may be performed to obtain specimens when diagnosisis difficult. Expectorated sputum. Three to five expectorated sputum specimenscollected in the early morning over a period of 3 to 5 days are usually adequatefor diagnosingthe respiratorymycobacterioses.Processingof additional sputumspecimens doesnot noticeablyimproverecovery.Poolingspecimens over a 24-h period is not recommended, sincepooledspecimens have beenassociatedwith increasedcontamination and recovery time (6, 98). Several expectoratedsputum specimenscollected a day or two after bronchoscopymay enhancedetection of mycobacteria. Sputum shouldoriginate from deepwithin the lung, and the patient shouldbe instructed carefully on how to cough up material resemblingan oyster. Although screeningfor upper respiratory tract contaminationis not necessary,there should be evidenceof lower-tract secretions(6,84). Each sputum or other respiratory secretion specimen should be 5 to 10 ml and may be collected or transferred to 50-ml, screw-cap,plastic, conical tubesfor transport to the laboratory. Induced sputum. Certain patients may have difficulty in producingsputumwithout assistance. In thesecases,warmed5 to 10% saline(or 10% glycerin and 15% saline) may be nebulized to inducecoughing.Caremustbe takenwith patients sufferingfrom certain respiratory illnessessuchas

6

CERNOCH ET AL.

CUMITECH

TABLE

3. Specimens for mycobacterial studies

Specimen

Respiratory Expectorated sputum Induced sputum Bronchoscopy fluid

Method

Comments

Early morning cough, “oyster” Nebulization (5-10% saline or 10% glycerin and 15% saline) Bronchoscopic aspiration of bronchial secretions or instilled-fluid wash

Sinus tract Swab Tissue Topical (skin granulation) Normally sterile Gastrointestinal Gastric

Stool Urine

Sterile body fluids Blood CSF, pericardial, synovial Peritoneal, bile, paracentesis, peritoneal dialysis effluent, pleural Other exudates

16A

(quantity)

One/day over 3-5 days; pooling over 24 h not recommended (5-10 ml) Label as “induced” (5-10 ml) Expectorated sputum 1 or 2 days after bronchoscopic specimen may be helpful (5-10 ml or more if available) Needle aspiration, biopsy; avoid swabs if possible Avoid if at all possible; use polyester if absolutely necessary, and submit in 7H9 broth media.

Biopsy (excision) Biopsy (excision or needle)

As available

Early morning prior to stomach emptying with nasogastric tube

Perform only if necessary. Use 3 separate samples on 3 consecutive days (stomach aspirate + 20-30 ml of instilled lavage fluid). Neutralize with 100 mg of sodium bicarbonate if not processed within 4 h Walnut-size sample (l-2 g) One/day over 3-5 days (X5 ml; entire single, first morning voided specimen preferred). Pooling over 24 h not recommended

Fresh stool Clean genitalia; first morning voided urine

BACTEC, Septi-Chek, or Isolator Appropriate tap and aspiration by syringe and needle Appropriate tap and aspiration or accumulation of fluid

10 -20 ml/culture At least one, and more if available (“2 ml up to 10 ml) At least one, and more if available (215 ml up to 50 ml if available)

Aspiration (avoid swabs if possible)

3-5 ml

asthma.To avoid nausea,collection is best attempted at least 2 h after a meal. The patient shouldbe coachedto breathe deeply through the mouth and to coughwhile covering the mouth to decrease possible aerosolization. Specimens shouldbe labeled“induced sputum” (6, 98). Gastric aspirate. Aspiration of swallowedsputum from the stomach is reserved for patients unable to provide sputum by other means.The procedureshouldbe performed early in the morning prior to any food intake. Gastric contentsare collected with a suction syringe connected to a tube insertedinto the stomach.Sterile saline(20 to 30 ml) maythen be introducedinto the stomach and aspiratedaslavagefluid. Both specimensmay be pooled in a sterile container. Three separate aspiratescollectedon consecutivedaysare recommended.Gastric aspirationsshouldbe processed rapidly (within 4 h), or 100 mg of sodiumbicar-

bonate shouldbe added asa buffer to neutralize anyacid,which is detrimentalto mycobacteria(6). Other specimens.Invasive collection techniquesmay be necessaryto diagnosepulmonary mycobacteriosisin somepatients.Transbronchial and brush biopsy specimensand bronchial alveolar lavage fluid may be collected during a bronchoscopicprocedure. In some difficult patients, computerized tomography-guided transthoracic needle biopsiesor open lung biopsiesmay be performed,with the latter method havingthe best yield. The precise method of collection should alwaysbe noted on the requisition. Urine Genitalia shouldbe appropriatelycleansedpreceding collection of first morning voided urine specimens. A minimumspecimenvolume of 15ml is acceptable, but the entire voided volume is

CUMITIXH

16A

DIAGNOSIS

OF THE MYCOBACTERIOSES

7

ulated into a BACTEC mycobacterial culture systemor a Septi-Chekmycobacterialculture system (Becton Dickinson) and processedfor inoculation to routine mycobacterial media (6, 98). Direct inoculation of blood into a BACTEC 13A vial is an acceptablealternative. Blood sediments Swabs from the Isolator tube shouldnot be inoculatedto Avoid collecting specimens with swabs whenever BACTEC 12B medium,sincethey may be inhibpossible. The hydrophobicity of the mycobacteria, itory to MAC organisms(129). which is due to their highly lipid-rich cell walls, often compromisestheir releasefrom suchswabs Body fluids and enhancesovergrowth with contaminatingorA variety of body fluids may be collected by ganisms.If swab-collectedspecimens are unavoid- asepticaspirationthrough a needleinto a syringe. able (e.g., laryngealswabs),they shouldbe trans- Alternatively, some fluids (e.g., effluent from ported in approximately 10 ml of Middlebrook chronic ambulatory peritoneal dialysis) may be 7H9 broth in sterile polypropylenetubes (6, 98). submitteddirectly in sterile collection bags.In all cases,appropriate volumesshouldbe transferred to sterile, plastic, conical, screw-captubes for Stool Stool specimensare recommendedfor detec- further processing. tion of MAC only in gastrointestinaltracts of Specimenscollectedin this mannermay include patients with AIDS. Stool specimensshould be cerebrospinalfluid (CSF); pericardial or synovial submittedin clean containers;swabsare not ac- fluids; peritoneal, bile, paracentesis,or pleural ceptable. A controversial recommendationsug- fluids; exudates,and peritoneal dialysiseffluent. gestsculturing stool only if the patient’s smears Because certain body fluids (such as CSF or showAFB. If cultured, a pea-sizedpiece of stool peritoneal dialysiseffIuent) may contain minimal should be emulsifiedin 5 ml of Dubos albumin numbersof mycobacteria,largespecimenvolumes broth, incubated overnight at 35°C and then increaseculture yield. Body fluids may be diluted in salineor a buffer processedlike a sputumsample. to allow the relatively buoyant AFB to sediment Normally Sterile Specimens adequatelyduring centrifugation (6, 98). Normally sterile specimensare usually considered devoid of contaminatingbacterial flora but Tissue (biopsy) may at times be contaminatedby organismsacSurgicalbiopsy tissueor other tissueshouldbe quired during collection. Specimensshould be submitted in sterile nonbacteriostatic saline or decontaminatedonly if the specimenis thought to Middlebrook 7H9 broth to prevent dehydration. be contaminated,sincesuch proceduresmay re- Large pieces of tissue should be cut up and duce the viable count of mycobacteria, thereby homogenizedin a mechanizedtissuegrinder or diminishingthe yield of culture. If bacterial con- ground in a mortar and pestlewith sterile sandor tamination is suspected,the specimenmay be Alundum powder. Specimensthat cannot be prorefrigerateduntil the routine bacterialculturesare cessedimmediatelymay be frozen at -20°C and examinedthe following day. If routine bacterial then transportedon dry ice (6, 98). culturesare not performed, the specimenmay be SPECIMEN PROCESSING inoculatedonto blood or chocolateagar platesto Specimenscollected from normally sterile sites test for bacterial contaminationbefore being processedfor mycobacteria(6, 98). are inoculated to media directly or centrifuged before inoculation. These include spinal fluid, joint fluids, and biopsy tissues(i.e., liver, heart, Blood Isolation of mycobacteriafrom blood ispossible spleen,etc.). Tissuesare maceratedwith a tissue primarily in immunocompromisedpatients, and grinder or stomacher. Large volumes of body such attempts should be reservedfor these pa- fluids (>lO ml), such as pleural fluid, should be tients (e.g., those with AIDS). Blood may be centrifugedbefore inoculation. Specimensthat are likely to contain normal or collectedin the Isolator lysis-centrifugationblood culture system(WampoleLaboratories,Cranbury, transientbacterial flora must be decontaminated. N.J.). The blood should remain in the Isolator Biopsy samplesof necrotic material or specimens tube for 1 h to allow completelysisof phagocytic that are colonized with bacteria should be procellsandto kill any HIV presentprior to process- cessedto kill the bacterial flora that is present. ing. Alternatively, blood can be initially collected Autopsy specimensmust always be decontamiinto a sterile glassVacutainer with sodiumpoly- nated. Processingof specimensat different instianetholesulfonate(SPS)asan anticoagulant(Bec- tutions may vary on the basis of nosocomial ton Dickinson,Cockeysville,Md.) and then inoc- problems and patient type. Common senseis preferable.Three to five specimenscollectedover asmanydaysare normally adequate.Twenty-fourhour pooled specimensare not recommended,as recovery of AFB may be compromisedby longterm exposureto urine.

8

CERNOCH ET AL.

CUMITECH

TABLE

4. Digestion and concentration of specimens”

Agent

NALC + 2% NaOH

Dithiothreitol (Sputolysin; Calbiochem, La Jolla, Calif.) + 2% NaOH 13% trisodium phosphate + benzalkonium chloride (Zephiran)

Comments

Mild decontamination with mucolytic agent NALC to free mycobacteria entrapped in mucus. NaOH may have to be increased to 3% to control contamination on occasion. NALC should be discarded after 24-48 h. Very effective mucolytic agent used with 2% NaOH. More expensive than NALC but has same advantages as NALC. Preferred by laboratories that cannot always control time of exposure to decontamination solution. Benzalkonium chloride should be neutralized with lecithin if not inoculated to egg-based culture medium. Avoid using with BACTEC

4% NaOH

4% Sulfuric acid 5% Oxalic acid

16A

system.

Traditional decontamination and concentration solution. Time of exposure must be carefully controlled. NaOH at 4% will effect mucolytic action to promote concentration by centrifugation. Use when decontaminating urine specimens has improved recovery for many laboratories. Most useful in processing of specimens that contain P. aeruginosa as contaminant.

a Modified from reference 110.

usuallythe bestguide for specimendecontamination. DIGESTION AND DECONTAMINATION METHODS Most specimens receivedfor mycobacterialculture contain various amountsof organic debris and a variety of contaminating,normal, or transientbacterial flora. A chemicaldecontamination processusually effectively kills the contaminants while allowing recovery of the mycobacteria.The high lipid content of the APB cell wall makesthe mycobacteria more resistant to both acid and alkalinedecontaminatingagents.Strict adherence to the timed killing period is necessaryto maximize recovery. The type of decontaminatingprocedure selectedshould reflect the number and type of specimensreceivedand the time and staff required to processthem. Some proceduresare better for hospitalizedpatients; others are better for outpatients.SeeTable 4 for a summaryof the agentsused. An acceptablecontaminationrate of specimens cultured on nonselectivemedia is 2 to 5% (58, 117).If the rate is higher than 5%, the decontamination procedure being used is inadequate. A higher concentration of the decontaminatingsolution might be consideredrather than a longer exposuretime for the specimen.If the rate is less than 2%, the procedure is too harsh and might damagethe APB; thus, the concentration of the decontaminatingsolutionshouldbe decreased.A review of the contamination rate of cultures should be conducted on a regular basis,either monthly or quarterly, and the proceduresshould be adjustedaccordingly.

NALC-NaOH Procedure Probably the most popular digestion and decontaminationprocedure in the United Statesis the N-acetyl-L-cysteine-2% sodium hydroxide (NALC-NaOH) procedure. A variety of specimens may be processedsuccessfullywith this method. NALC acts asa mucolytic agent,breaking the disulfide bondsin the specimenand resulting in liquefaction (67). NALC quickly loses its mucolytic activity and must be usedwithin 24 to 48 h once it is in solution. If the specimen is extremely viscous,more NALC may be added to help with liquefaction. Sodium hydroxide is the decontaminatingagent, and the concentration may need to be increased during warm weather or in casesof persistentcontamination. Sodiumcitrate aidsin the prevention of inactivation of the NALC by binding metal ions that can be present in solution (70). The phosphatebuffer used to dilute the decontaminatingsolution decreasesthe specificgravity to makesedimentation of the APB more effective. Adding 0.2% albumin to the sedimentafter centrifugation results in a buffering and detoxifying effect on the sediment. 4% SodiumHydroxide Petroffs 4% sodium hydroxide procedure requiresvery strict adherenceto the timed decontaminating step becauseof the toxicity of this agent to mycobacteria (58). Sodium hydroxide actsasthe digestantaswell asthe decontaminating agent.

CUMITEXH

16A TABLE

DIAGNOSIS

OF THE MYCOBACTERIOSES

9

5. Commercial availability of common media and reagents for mycobacteriology Medium

or reagent

Company”

American Trudeau Society .......................................................................................................... BBL, Remel, C-S Arylsulfatase broth, 3-day test .................................................................................................... BBL, Remel Arylsulfatase broth, 14-day test .................................................................................................. BBL, Remel Arylsulfatase butt .......................................................................................................................... BBL, Remel Dubos Tween albumin broth ...................................................................................................... BBL, Remel, C-S LJ slant .......................................................................................................................................... BBL, Remel, C-S, Adams LJ butt ............................................................................................................................................ BBL, Remel, C-S, Adams LJ with 5% NaCl .......................................................................................................................... BBL, Remel, C-S LJ Gruft (with penicillin and nalidixic acid) ............................................................................. BBL, Remel, C-S LJ Mycobactosel (with cycloheximide, lincomycin, and nalidixic acid) slant ....................... BBL, Remel Middlebrook 7H9 broth with tween 80 ..................................................................................... BBL, Remel, C-S, Adams Middlebrook 7HlO slant and plate ............................................................................................ BBL, Remel, C-S, Adams Middlebrook 7Hll slant and plate ............................................................................................ BBL, Remel, C-S, Adams Middlebrook 7Hll plate with mycobactin J ............................................................................. Remel Nitrate substrate bro&h ................................................................................................................ BBL, Remel, Difco Pyrazinamidase butt ..................................................................................................................... BBL, Remel TCH slant ...................................................................................................................................... BBL, Remel Tellurite reduction broth ............................................................................................................. BBL, Remel Urea broth ..................................................................................................................................... BBL, Remel Phenolphthalein disulfate tripotassium salt .............................................................................. Sigma Tween 80, 10% solution .............................................................................................................. Remel Hydrogen peroxide, 30%, superoxol .......................................................................................... Sigma Tween 80 hydrolysis concentrate ................................................................................................ Remel Ferric ammonium citrate, 20% ................................................................................................... Remel Niacin strips ................................................................................................................................... Difco, Remel Potassium tellurite (0.2%) ........................................................................................................... Remel N-acetyl+cysteine’ ...................................................................................................................... Remel, Sigma, Adams OADC enrichment ....................................................................................................................... Remel, Difco Dithiothreitol (Sputagest 50) ...................................................................................................... Remel TB base digestant (NaOH-Na citrate)’ ..................................................................................... Remel, Adams Benzalkonium chloride ................................................................................................................ Sigma Neutralizing buffer ........................................................................................................................ Difco Sodium bicarbonate ...................................................................................................................... Sigma u BBL, Cockeysville, Md.; Remel, Lenexa, Kans.; C-S, Carr-Scarborough, Decatur, Ga.; Adams Scientific, West Warwick, R.I.; Difco, Detroit, Mich.; Sigma Chemical Co., St. Louis, MO. b Complete kits containing all materials necessary for decontamination and digestion with NALC-NaOH are available from various manufacturers, including Remel and Alpha-Tee, Irvine, Calif.

Dithiothreitol with SodiumHydroxide Dithiothreitol, or Cleland’sreagent, is another mucolytic agent (105). The compound is more stablein air than NALC but ismore expensiveand breaksthe disulfide bondsin the sameway that cysteinedoes.

basedmedia, lecithin must be used as a neutralizer (62). This systemcannot be usedin conjunction with the BACTEC system.

Other Procedures The 6% oxalic acid procedure is useful with specimens that are consistentlycontaminatedwith 13%Trisodium Phosphateand Benzalkonium Pseudomonas aeruginosa. Sulfuric acid (4%) is Chloride usually used only for urine specimens.Sodium Trisodium phosphate (13%)-benzalkonium lauryl sulfate and cetylpyridinium chloride (108) chlorideis a gentlerdecontaminatingreagentthan are seldomusedany more and are incompatible sodium hydroxide. This systemshould be used with the BACTEC system. whenthe laboratory is unableto monitor the time of exposureto the decontaminatingagentor when MEDIA the specimensare particularly bloody. BenzalkoNonselective nium chloride (Zephiran) is the decontaminating agent,and trisodiumphosphateacts asthe digesCulture mediausedin the recovery of mycobactant. The sedimentshouldbe inoculated to egg- teria are divided into three groups: broth, egg basedmedia, in which the phospholipidsact as a based,and agarbased(Table 5). A variety of each built-in neutralizer for the benzalkoniumchloride type of medium should be used in the primary (24). If the sedimentis to be inoculated to agar- isolationof AFB.

10

CERNOCH

ET AL.

CUMITECH

16~4

Draft guidelines developed by the CDC (20) for and observethe microcolonies,thusallowingearly isolating M. tuberculosis state that patient speci- identification of M. tuberculosis and other species. mensshouldbe processedand placed in a broth Middlebrook 7HlO and 7Hll are commonlyused and a solid medium, as broth media promote a for primary isolation and drug susceptibilitytestmore rapid positive result. Two broth systems ing. The addition of 0.1% caseinhydrolysategives (BACTEC and Septi-Chek) are commercially 7Hll an improvedrecovery rate for drug-resistant available. Laboratories that use the BACTEC mycobacteria(31). Both 7HlO and 7Hll contain systemusuallyalsoinclude an egg- or agar-based malachite green as a bacterial inhibitor but at a medium. The Septi-Chek system incorporates much lower concentration than that usedin eggboth broth and solid media. basedmedia.Five to 10% CO, is essentialfor the Middlebrook 7H9 and Dubos Tween albumin growth of mycobacteriaon both 7HlO and 7Hll. broths are the broths most commonly used for Care must be taken to keep Middlebrook 7HlO subculturingand preparing inocula for drug sus- and 7Hll from exposureto sunlightor fluorescent ceptibility tests.They can alsobe usedfor recov- light, asdeterioration of the mediumand release ering small numbersof organismsfrom sterile of formaldehyde will result. Storage at 4°C for specimenssuch as CSF in the absenceof the more than 4 weekswill have the sameeffect. BACTEC or Septi-Cheksystem.The addition of Selective Tween 80 to the mediumallowsfor homogenous growth and dispersalof the clumpsof mycobacteThe recovery of mycobacteria may be greatly rial growth. enhancedby the use of selective media. Both Ingredientsof egg-basedmedia include whole egg-basedand agar-basedmedia may be made eggsor yolks, potato flour, salts,asparagine,and selectiveby the addition of antimicrobial agents. glycerol;thesemediaare solidifiedby inspissation. The Gruft modificationof LJ is probably the most Egg-basedmedia usually resist drying for pro- widely used of the selective egg-basedformulalongedincubationperiods;this providesan advan- tions;penicillin and nalidixic acidare addedto the tage over agar-basedmedia. Additionally, egg- LJ base,which alsocontainsRNA. Various conbased media support good growth of most centrationsof lincomycin, cycloheximide,and namycobacteriaand provide a greater yield of posi- lidixic acid can be addedto either LJ (Mycobactive cultures (63). Egg-basedmedia also allow tosel; BBL, Baltimore, Md.) or 7HlO (95). tracesof toxic materialsto be neutralized. MalaSelective 7Hll (7HllS) is a modification of chite green is added as a bacterial inhibitor in Mitchison’s original medium made by reducing mostegg-basedmedia.Aniline dyes,suchascrys- the concentration of carbenicillin and changing tal violet, may alsobe usedasbacterial inhibitors, the agarbasefrom 7HlO to 7Hll. Using7HllS in but their concentrationsmust be low, because addition to LJ and 7Hll has resulted in an aniline dyesmay alsoinhibit the growth of myco- increasedrecovery rate of mycobacteriaover that bacteria. A disadvantageof egg-basedmedia is obtainedwith nonselectivemedia(79). Originally, that when contamination occurs,the entire sur- 7HllS wasdesignedto isolatemycobacteriawithface of the medium may be affected, and ulti- out the use of a decontaminating procedure; mately, the mediummay completelyliquefy. Egg- however, by using 7HllS in conjunction with a based media are not commonly used in drug decontaminating process, in particular, the susceptibilitytestsbecauseegg fractions may in- NALC-NaOH procedure, the rate of recovery is terfere with somedrugs. improved. The most commonlyusedegg-based mediumis Selective culture media should not be used Lowenstein-Jensen medium(LJ). Petragnanime- alonefor the recovery of AFB but shouldbe used dium hasan increasedconcentrationof malachite with a broth-basedmediumor nonselectiveagargreen,which favors its usefor contaminatedspec- basedor egg-basedmedium. imens,such aswounds or abscesses. Growth on BACTEC System this medium may be slower than that on LJ. American Trudeau Society medium has a much A radiometric detection system that uses a lower concentration of malachite green and is mediumcontainingpalmitic acid labeledwith 14C indicated for normally sterile specimenssuch as was developedby Middlebrook and coworkersin CSFandjoint fluids.A simplemodificationof LJ, 1977(80). The mycobacteriacatabolize the 14Ci.e., adding0.4% pyruvate, enhancesthe recovery labeled palmitic acid and release 14C02; the of M. bovis (35). 14C02 in the headspacegas is measuredby an Agar-basedMiddlebrook and Cohenmediaare automatedinstrument (BACTEC 460) developed transparentmediathat allowdetection of colonies by Becton Dickinson Instrument Systems,Sparks, at 10to 12days,whereasopaquemediasuchasLJ Md. The 14C0, releasedis measuredquantitausually exhibit colonies at 18 to 24 days. The tively on a scalefrom 0 to 999.The actualnumber transparencyof the mediumallowsthe technolo- is designatedthe growth index, or GI reading.Any gist to invert the plate on a dissectingmicroscope increasein the GI is directlv proportional to the

CUMITECH

16A

rate of growth of APB in the medium.A Middlebrook 7H12 mediumis usedwith or without the addition of PANTA, an antibiotic solution containing polymyxin B, azlocillin, nalidixic acid, trimethoprim, and amphotericin B. Middlebrook 7H12 mediumis a 7H9 broth basewith catalase, albumin, and caseinhydrolysateadded. BACTEC vials are readtwo or three timeseach weekfor the first 2 to 3 weeksand oncea week for the remaining culture time. Detection time for growth of positive cultures in BACTEC broth mediumis decreased(comparedto that on solid medium)to lessthan 7 daysfor someNTMs and to 4 to 25 daysfor M tuberculosis. Rapid identification of M. tuberculosis may alsobe performed in the BACTEC systemby usingthe NAP (p-nitroacetylamino$-hydroxypropiophenone)test. Susceptibility testing for M. tuberculosis may alsobe performed in the BACTEC systemand is completed in 10 daysor less,comparedto 3 weekson solidmedia. BACTEC 13A mediumis usedwith blood and bone marrow aspirates.This mediumis BACTEC 12B broth with the anticoagulantSPSadded.The volume of the mediumis 30 ml, unlike the 4-ml volume of the 12B vial, and therefore allows a 5-mlvolume of blood to be addedto the vial. The blood is inoculateddirectly into the mediumat the patient’s bedside or collected in a yellow-top Vacutainer with SPSand subsequentlytransferred to the blood culture vial. A supplementcontaining bovine serumalbumin is addedto the 13A bottle when it is received in the laboratory. Septi-ChekAFB RocheLaboratoriesoriginally developeda system (Septi-ChekAPB) for the isolation of mycobacteria that is now marketedby Becton DickinsonMicrobiology Systems.The systeminvolves a mycobacterialbroth culture bottle, a mycobacterial agar slide, and a mycobacterialculture supplement. The culture bottle contains 20 ml of modified Middlebrook 7H9 broth in a CO,-enriched (20 to 40%) atmosphere.The mycobacterial slide has three media: nonselectiveMiddlebrook 7Hl1, LJ, and chocolate agar. Bacterial contaminantsshouldbe evident on the chocolate agar. The culture supplementcontains glycerol, catalase,albumin,and severalantibiotics(azlocillin, nalidixic acid,trimethoprim, polymyxin B, and amphotericinB). The specimenis inoculatedinto the culture vial, and the slide is attached. Once the specimenis inoculated,the bottle is turned upsidedown and incubated in a vertical position. The systemrequiresdaily and then weekly bottle inversionuntil coloniesare seen.Both the broth and the solid mediashouldbe observedfor growth and stained for APB when growth is detected. This system usually yields APB several days later than the

DIAGNOSIS

OF THE MYCOBACTERIOSES

11

BACTEC system,although the overall isolation rate approachesthat of the BACTEC (104). INCUBATION CONDITIONS Media used for primary isolation should be incubated in 8 to 10% CO2 for at least 1 week. Growth on Middlebrook agar requires CO, to enhance growth, whereasgrowth on egg-based media is stimulatedby the increasedconcentration of COZ.Candleextinction jars are unacceptablefor usein the mycobacteriologylaboratory, as they produce only 3% CO,. Agar-basedmediain platesshouldbe incubated medium side down in CO,-permeablepolyethylenebags.The numberof platesin eachbagshould be limited to allow adequatecirculation of CO2 and to minimizewater condensationon the plate lids. Alternatively, platesmay be taped individually with gas-permeable tape. LJ tubesshouldbe slantedfor 1 week to allow better CO2 perfusion of the slant and then may be placedupright. Lids shouldbe loosenedto allow CO, exchange. Culturesshouldbe routinely incubatedat 35 to 37°C with high humidity. Additionally, skin and soft tissuecultures, cutaneouslesioncultures, or M. haemophilum culturesof isolatesfrom noncutaneoussitesshouldbe incubated at 30 to 33°C since organismssuch as M. marinum, M. haemophilum, M. chelonae, and M. ulcerans have lower optimal-temperature requirements and sincemany of theseisolatesgrow poorly or not at all at 35 to 37°C on primary isolation.M. avium andM. xenopi exhibit optimumgrowth at 42OC.It is important that the 12B and 13A BACTEC vials be incubated at 37 t l”C, as incubation at temperaturesbelow 36°C increasethe detection time of a positive culture. Cultures on solid medium shouldbe examined within 1 weekof inoculationandweekly thereafter for a total of 6 to 8 weeks.BACTEC vials should be read two or three timesa weekfor the first 2 to 3 weeks and weekly thereafter for a total of 6 weeks.If a conventional liquid medium (Middlebrook 7H9 or Dubos) is usedfor primary isolation, carefully inspectthe mediumfor particulate matter and stain before resultsare finalized. A hand lensmay be usedfor examiningopaque media. Using a dissectingscopefor agar-based media also aids in the early detection of microcolonies.Some laboratories reincubate the agar platesfollowing initial isolationof acid-fastorganismsin order to seeif a secondspeciesof APB will grow. MICROSCOPY Mycobacteria have distinct and characteristic cell wallscomposedof complexfatty acidscalled mycolic acids.Thesecharacteristiccell walls distinguish mycobacteria from other microorganisms. In fact, the term APB derives from the

12

CERNOCH

ET AL.

dye-absorbing properties of these mycolic acids that are resistant to acid-alcohol destaining procedures. This property provides a simple, rapid, inexpensive means of presumptively diagnosing new cases of active M. tuberculosis infections.Over the last 20 years, the most commonAFB smear methodshave proven to be reasonablysensitive and specific compared to conventional culture isolationsand biochemicalidentification methods. However, definitive identification still requires growth and biochemicaltesting. Besidesallowing presumptive identification of M. tuberculosis and/or other mycobacterioses,AFB smearsprovide additionalusefulinformation. Growth recovered from sputum,sediments,or other specimen sourcescan be confirmed by an AFB smear.The semiquantitativeresultsof anAFB smearareused for establishingthe inoculum concentration for direct susceptibility tests. Smear results can be usedto determinethe adequacyof the specimen decontamination-sedimentation procedure. If too many specimensare smearpositive and culture negative, then the decontaminationstep is too harshor too long. Resultsobtained from smears made during the courseof the patient’s chemotherapy can be usedto evaluatethe successof the treatment and may even be used to determine whetherthe patient shouldbe dischargedfrom the hospital (66, 98, 110).

CUMITECH

MA

lution of malachitegreen. The procedure for the auramine0 fluorochromedye is similarto that of the carbol fuchsinmethod.The heat-fixedsmearis stained with an alcohol-basedsolution of auramine 0 (with or without rhodamine),rinsedwith water, decolorized with an acid-alcoholsolution, and rinsed with water again. The smearis counterstained with a potassiumpermanganatesolution, rinsedwith water, and air dried before being read. Fluorochrome-dyedsmearsshould be read the sameday that they are prepared,becausethe fluorescencebecomesweaker with time (58, 66). Microscopic

Examination

of Smears

Carbol fuchsin-stainedsmearsare examined with 100X oil immersionobjectives(approximately X 1,000magnification)usingbright-field microscopy. The CDC recommendsthat three parallel, longitudinal sweepsof the smears(i.e., approximately 100 fields per sweep,which is 300 fields total per smear) be examined carefully. This method requires about 15 min per slide. Fluorochrome-dyed smearsare read by fluorescence microscopy(mercury vapor lampor strong,appropriately filtered blue light) using X150 magnification for scanning and ~450 magnification for confirming AFB morphology. At the lower magnification, three parallel sweeps(minimum of 30 fieldsof view) shouldbe examined.The aurominerhodaminemethod is much quicker, and a smear Procedures can be read in as little as 1 to 2 min. Positive Three commonproceduresare widely usedfor fluorochromesmearscan be overstainedby one of AFB smears.Two methods, Ziehl-Neelsen and the carbolfuchsinmethodsto confirmAFB and to Kinyoun, usecarbol fuchsin asthe primary stain, store slidesfor future reference(58, 66). and in thesemethods,AFB are observedby light Interpretation of Results microscopy.The third method usesthe fluorochrome dye auramine0, and AFB are observed The recommendedinterpretations and reportby fluorescencemicroscopy.Each stainingproce- ing of smearresults by the ATS and CDC are dure uses the same basic sample preparation given in Table 6. If a final result indicates an method. For example,a sputum specimenor its indeterminatenumberof AFB seenon the smear, sedimentshouldfirst be vortexed to ensureade- a repeat smearfrom the samespecimencan be quate sampling.Two or three drops of the speci- made, and another specimenshould alsobe remenshouldbe carefully spreadonto an area (1 by quested(58). 2 cm) of a new, clean microscope slide and In general, the specificity of AFB smearsis allowedto air dry. The smearshouldthen be heat quite good; however, other organisms,such as fixed at 65 to 75°C on a slide warmer for 2 h or Nocardia, Rhodococcus, Corynebactetium, Cryptopassedthrough the blue cone of a bunsenburner sporidium, and Isospora spp.,are variably acid fast flame several times. Proceduresfor the carbol (58). It has been reported that up to 10% of fuchsin staining methodsare quite similar. The AFB-positive smearsare culture negative due to heat-fixed smear is stained with carbol fuchsin effective chemotherapy (110). These specimens reagent.Then, in the Ziehl-Neelsenmethod, the need to be evaluatedwith respectto the precaustainisheatedon the slide.The stainisnot heated tions discussedbelow. Sensitivity may be better in the Kinyoun method. For both methods,after with the fluorochrome stain than with the carbol the slideis stained,it is rinsedwith water, decolo- fuchsin stains. The sensitivity of AFB smears rized with an acid-alcohol solution, and again rangesbetween30 and 80% of culture isolations rinsedwith water. In the Ziehl-Neelsenmethod, and is dependenton both the culture isolationand the smearis counterstainedwith an aqueousso- recovery methodsusedandthe patient population lution of methyleneblue, rinsedwith water, and beingevaluated(110). In general,AFB smearsare air dried before being read. In the Kinyoun regardedashavinga sensitivityon the order of lo5 method, the counterstainis an alcohol-basedso- AFB per ml of sputum.In comparison,the sensi-

CUMITECH

16A

DIAGNOSIS TABLE

OF THE MYCOBACTERIOSES

13

6. Smear evaluation”

No. of AFB observed Fluorochrome Fuchsin stain, x 1,000

x450 RWS

0

l-2/3OOF (3 sweeps) l-9/100F l-9/10F 1-9/F >9/F

stain

0

l-2/30F (1 sweep) 109/10F 1-9/F 10-90/F >9o/F

0

-’ -b -b p

RWS 0

l-2/70F (1.5 sweeps) 2-18/50F (1 sweep) 4 -36/10F 4-36/F >36/F

Report

x630 RWS

0 C

C

c C C

0

0

l-2/130F (2 sweeps) 2-18/100F (1.5 sweeps) 2-18/10F 2-18/F >18/F

-d

No AFB seen Doubtful; repeat

-d

l+

-d d d

2+ 3+ 4+

a From reference 58. In all cases,one full sweep refers to scanning the full length (2 cm) of a smear 1 by 2 cm. K/K, Kent and Kubica; RWS, R. W. Smithwick; F, field. b -, Divide the observed count by 10. -, Divide the observed count by 4. d -, Divide the observed count by 2. C

tivity of culture is regardedas10to 100CFU/ml of sputum(58). A recent study showedthat useof a cytocentrifuge to prepare smearsfrom clinical specimens resultedin a significantimprovementin the detection of AFB (100). Precautions

Besides the fact that AFB smearsare less sensitivethan culture, severalfactors can contribute to false-negativeAFB smears.First, intermittent sheddingof tubercles (small nodules with caseouscenterscontainingdormant1M.tuberculosis) from patients or inappropriately induced or collected sputum samplescan result in negative smears.Second, inadequately destained or decolorized smearscan causefalse-negativeinterpretations. Third, smearsthat are too thick can reducethe likelihood of detectingAFB, especially with the fluorochromedye,which canbe quenched by too much material. Also, smearsthat are too thick might flake off the slide.Fourth, inadequate examinationof the smearby observing too few fieldscanresult in failing to observeAFB that are present(66, 109). Similarly, a number of factors can causefalsepositiveAFB smears.First, consistentoccurrence of more than 2% AFB-positive smearsand culture-negative results on all specimensindicates that someof the AFB smear reagentsor slide rinse water may be contaminated(58). Another possibilityisthat the 100X oil immersionobjective is being cross-contaminatedbetween slide readings; the objective should be carefully cleaned after eachpositive smear(6). Another possibility is that smearsthat are too thick flake off during processingand cross-contaminateother slides. Therefore, slidesshouldbe handledcarefully, and commonstain jars shouldnot be used.Alternatively, the decontaminationstep of the sediment

preparation may be too harsh,too long, or inadequately neutralized before culture inoculation. Last, AFB-positive smearsassociatedwith culture-negative specimenscan be due to effective chemotherapy treatment of the patient. Thus, previous laboratory results on a given patient shouldbe checked(58, 66, 109, 110). IDENTIFICATION

TECHNIQUES

Mycobacteria have traditionally beenidentified by usinga number of phenotypic properties such as growth rate, pigmentation, colonial morphology on translucentagar media,and, for definitive purposes,biochemicaltest characterization (98, 99) (Table 7). A battery of conventional testsfor the identification of mycobacteria has been developed and standardizedover the past several decades.The advantagesof these methods are that they are well-established,relatively inexpensive,standardized proceduresthat are in usein the majority of mycobacteriology laboratories. Their disadvantagesare that they require dedicated,experienced technologists;must dealwith multiple strain variations within species;and are slow in providing clinically relevant information, which, overall, probably increasespatient care and preventivetreatment costs. Conventionalbiochemicaltestsusedto identify mycobacteriainclude niacin accumulation,nitrate reduction, Tween 80 hydrolysis,iron uptake, inhibition of growth by thiophene-2-carboxylicacid hydrazide (TCH), ability to grow on MacConkey’s agar, tolerance of 5% sodiumchloride, and production of the enzymescatalase(both semiquantitative and heat stable at 68”C), arylsulfatase, pyrazinamidase,and urease.Details on the performance of the biochemical tests are not pre-

14

CERNOCH ET AL. TABLE

7. Distinctive properties of cultivable mycobacteria encountered in clinical specimens” Growth

rate at!‘:

Species M. M. M. M. M. MI M. M. M. M. M. M. M.

ulcerans tuberculosis bovis marinum kansasii simiae asia ticum scrofulaceum szulgai gordonae JEavescens xenopi avium-M. intracellulare

45°C

37°C

31°C

24°C

-

s S -/+ s S S s s S M s s

s s

M s

-

s -/+

M s s s

Usual colony morphology’

Ro Ro Rt Sm/SR SR.fSm Sm

gastn’ malmoense haemophilum nonchromogenicum ttwrae triviale fort&urn group’ chelonae-M. abscessus

group

M. phlei M. smegmatis M. vaccae

-

R R

S s S S M R R R R R

s Sh

Pigmentatio&

Niacin

(63) (0) (0) (0) (0) (0) (0) (0)

(0) (0)

-

- (0) - (1)

-

t + + -

-+

Sm Sm or Ro Sm Sm Smh St/R0

t --

S s s S S S R R

Sm/SR/Ro Sm Ro SR SR Ro Smf/Rof Sm/Ro

N N N N N N N N

-/+ -/+

R R R

Ro Ro/Sm Sm

S N S

sented here, as they have been thoroughly addressedin other papers(58, 98, 109). Newer methodshave been standardizedduring the past decade.Theseinclude the NAP test for identification of M. tuberculosis with the BACTEC system,chromatographicrecognition patterns of the mycobacteria cell wall fatty acids (mycolic acids),and rapid identification of the most clinically significantand prevalent speciesby nucleic acid probe assays.The advantagesof the last two methodsare that they are capableof providing definitive identificationlessthan 2 h after recovery of adequategrowth andthat both are more objective methods that can be readily mastered by technologists.Consequently,these methods are more cost-effectivein the overall managementof patients and the prevention of tuberculosis. Key BiochemicalTests Members of the M. tuberculosis complex (M. tuberculosis, M. bovis, M. africanurn, and M. microti, a wild rodent pathogenonly) are considered strictly pathogenic, are slow growers, and are nonpigmented.Coloniesshowtight cording (arrangementin “serpentine-cord”formation) and a distinctively patterned texture with a buff color. Key biochemicaltestsfor this group of mycobacteria includeproduction of niacin and heat-stable catalaseand reduction of nitrate.

(100) (88) (93) (100) (100) (100)

Nitrate reduction

+ (97) -- (9 (0) +- (99) - (28) - (5) (5) +- w9 (1) (92) -+ (7) - (4)

+- (95) (4) -/+ (21) - (4)

S s s S M

Susceptibility to T2H (5 l4w)

+-

-

N

N (100) N WQ P (100) p (96) p PO) p (86) s (97) s/p (93) s (99 s (100)” s (21) N (87)

complex M. M. M. M. M. M. M. M.

16A

CUMITECH

(1) (0)

-

(67) (89 (100) (1)

+ +

There is presently somedebateconcerningthe status of the speciesM. africanurn, which, from biochemicaland DNA relatednessstudies,seems indistinct from M. bovis (43). M. tuberculosis may be separatedfrom M. bovis by the latter’s susceptibility to TCH (84, 98, 109, 110). The following key biochemicalprofiles (6, 43, 84, 109, 110) may be useful in differentiating the NTM to specieslevel. Photochromogens:nitrate reduction, Tween 80 hydrolysis,semiquantitativecatalase,and urease Scotochromogens: nitrate reduction, Tween 80 hydrolysis, urease,and tolerance of 5% sodium chloride Nonphotochromogens:nitrate and tellurite reduction, semiquantitative and heat-stable catalase,and Tween 80 hydrolysis Rapid growers:arylsulfatase,nitrate reduction, iron uptake, and citrate For clinical purposes,biochemicallysimilarspecies of NTM may be grouped as complexes. Further differentiation of isolateswithin these groupsis not usuallynecessary.Thus, sincedifferentiatingbetweenM. avium andM. intracellulare is difficult with biochemicaltests,it is appropriateto consider the two as a single complex (MAC). However, the previously designatedM. fortuitum complex shouldnot be usedto group the rapidly

CUMITECH

16A

DIAGNOSIS TABLE

Semiquantitative catalase (mm of bubbles)

~45 ~45 ~45 >45 >45 >45 >45 >45 >45 >45 >45 >45

45 (89) (69) (98) (93) (93) (95) (84) (98) (90) (94) (85) (98)

>45 (100) >45 (99) c45 <45 <45 (93) <45 (100) <45 (93) >45 (92) >45 >45 >45

68°C catalase

Tween hydrolysis

+ - (1) k - (2) - (30) + + (91) ++ (9% + (95) ++ (94) + (93) -/+ + + (96) + (100) t (31) If: (60)

(68) (21) (97) (99) (9) (95) (2) (49) (100) + (100) - (12) - (2)

- (11) 2 (66) -

+ (100) +- (99

+ + (92)

+ + (99

+ (100) + (100) + (90) -/+ (43) 2 (53) -/+ (39)

+ + +

+ + +

Tellurite reduction

Tolerance to 5% NaCl

t -

Iron uptake

(0) (0) (0) (0) (0) (0) (62) (0) (0)

Arylsulfatase, 3 days

-

-

- (0) (0)

-I

,5 v v v-

(41Y (0) (0) (0) (0) (0) (0) (0) t (36) - (1)

-/+ (46) - (2)

- (25) + (100) + (92) + (85) + (89) vi

+ f

MacConkeY agar

-/+

-

5 (50) - (0) + (74) - (0)

+ + +

15

7. Continued.

-/+ (36) - (0) - (0) -I+ (39) - (0) -/+ (31) + (82) - (20) t (64) t (53) - (29 -/+ (44) t (65) + (81)

OF THE MYCOBACTERIOSES

-

-

- (2)

Pyrazinamidase, 4 days

ureaSee

v

Nucleic acid probes available

-

t (64) 2 (50) + (83) -/+ (49) 2 (69) - (10) v (31) + (72) v (31) + (72) - (0) - (2)

+ + + iI + -/+ + v +

+ + + + +

-/+ (44) - (9)

+

-

+

-

v

-

v

-

-I+ + +

v v + +

-

+ -

t (56) + (97) + (95)

v + +

+ + +

-

-

(13) (33) (70) (89)

u Modified from reference 98. Plus and minus signs indicate the presence and absence, respectively, of the feature; blank spaces indicate either that the information is not currently available or that the property is unimportant. V, variable; t , usually present; -/+, usually absent. Percentage of CDC-tested strains positive in each test is given in parentheses, and test result is based on these percentages. MAIS group is scotochromogenic, Tween negative, nitrate ne ative, catalase less than 45 mm, and urease positive (rarely); tellurite gives inconsistent results. %S, slow; M, moderate; R, rapid. c Ro, rough; Sm, smooth; SR, intermediate in roughness; t, thin or transparent; f, filamentous extensions. d P, photochromogenic; S, scotochromogenic; N, nonchromogenic. M: szui’gai is scotochromogenic at 37°C and photochromogenic at 24OC. e Urease test was performed by the method of Steadham (111). f Reaction at 14 days is positive. g Young cultures may be nonchromogenic or may possess only pale pigment that may intensify with age. h Requires hemin as growth factor. ’ Includes M fort&urn, M peregrinum, and M fort&urn third-biovariant complex. J M. chelonae is negative; M. abscessus is positive.

growingM fortuitum group, M. abscessus, and M. chelonae, becausethese three are easily differen-

ized by high MICs of cefoxitin (~64 @ml) and susceptibilityto tobramycin (MIC of 54 @ml). tiated biochemicallyand have significantlydiffer- In contrast, M. abscessus isolatesrequire lower ent drug susceptibilityprofiles (43, 109, 113). MICs of cefoxitin (~64 pg/ml) and are resistantto Wallace (125) has reported that susceptibility tobramycin (MIC of >8 pg/ml) (121). profiles of clinically significant rapidly growing speciesmay be useful in differentiating the M. Test Methods fort&urn group (M. fort&urn, M. peregrinum, and the third biovariant unnamedcomplex) from the Colonial morphology M. chelonae group (M. chelonae andM. abscessus). Colonial morphology (Table 8) of individual Members of the M. fortuitum group are usually coloniesshouldbe observedby usinga hand lens susceptibleto ciprofloxacin, ofloxacin, polymyxin or a dissectingmicroscope.An acid-fast stain B, sulfonamides,doxycycline, and pipemidic acid, shouldbe performed to make surethat the colowhile isolatesof M. abscessus andM. chelonae are nies are truly acid fast. Someisolatesmay show resistantto theseagents.M. chelonae is character- more characteristiccolony morphology on agar-

16

CERNOCH ET AL. TABLE Organism

M. avium-M.

intracellulare

M. bovis

M. chelonae-M.

abscessus

CUMITECH

8. Growth characteristics of commonly isolated mycobacteria” Growth (days)

group

Buff to yellow

25-90

Colorless

Colorless

3-7 3-7

lo-14

M. gastri

lo-21

M. haemophilum

14-28

M. kunsasii

IO-21

M. malmoense

28-42

M. scrofilaceum

Dark

Buff to yellow

M. gordonae

M. marinum

Pigment production Light

lo-21

group M. fortuitum

16A

5-14 lo-14

Colony morphology

on LJ

Usually smooth, dome shaped, and buff; rough, wrinkled colonies sometimes seen; cultures sometimes appear impure since more than one colony type may appear on same culture Low, smooth, colorless, pyramid shaped

Buff

Buff

Usually rounded, smooth, colorless, and hemispheric; rough colonies occasionally seen, especially on prolonged incubation Buff Soft, butyrous, hemispheric, and multilobate or Buff rough with heaped centers, although nonpigmented, may appear green owing to absorption of malachite green Round, smooth, convex, yellow to orange, and Yellow to Yellow to orange glistening orange Colorless to Colorless to Round, smooth, convex, and glistening; often buff buff resemble MAC colonies Buff to gray Buff to gray Nonpigmented, predominantly rough with smooth variants occurring frequently; media must be supplemented with ferric ammonium citrate or hemin Yellow Buff Smooth or rough; although nonpigmented when grown in dark, become lemon-yellow when exposed to light for 1 h, on continuous light exposure, characteristic orange-red crystals of beta-carotene appear

Buff

Buff

Growth observed sometimes only after 6 wk of incubation; colonies dysgonic, smooth, and colorless even after light exposure

Yellow

Buff

Similar to those of M. kansasii

Yelloworange Yellow

Yelloworange Buff

Smooth, moist, yellow-orange, and round

M. simiae

7-14

M. szulgai

14-28

Yellow to orange

Buff at 25°C; yellow at 37OC

M. tuberculosis

12-28

Buff

Buff

M. xenopi

28 - 42 Yelloworange

Yelloworange

Smooth; although nonpigmented when grown in dark, became yellow-orange when exposed to light Smooth to rough, pyramid shaped, with somewhat irregular periphery; scotochromogenic (orange) when incubated at 37OC and photochromogenic at 25°C; continuous light exposure may result in formation of red crystals Nonpigmented, dry, rough, with nodular surface and irregular thin periphery Small, smooth, dysgonic, dome shaped, yelloworange

basedmedia than on egg-basedmedia. The ap- viewed with transmittedlight is useful in identifipearanceof colonialmorphologyon plates(incu- cation (98, 99). Coloniesare often characterized bated for 5 to 14 days) inverted under the low- as smooth or rough, thin or transparent, and power objective (10X) of a stereomicroscope and filamentousor nonfilamentous(109). For exam-

16A

CUMITFXH

DIAGNOSIS TABLE

Colony morphology

on 7HlO agar

OF THE MYCOBACTERIOSES

17

8. Continued. Features in Middlebrook

7H9 broth

Thin, transparent, glistening or matte, smooth, entire and rounded; some are rough and wrinkled; “pitted” centers may be seen

Uniformly homogeneous suspension

Small, thin, often nonpigmented, raised, rough, later wrinkled and dry; some are inhibited on this medium As on LJ

Heterogeneous, finely granular suspension

l- to 2-day-old colonies show branching filaments, although older colonies do not; on cornmeal agar, branching filaments are conspicuous on both young and old colonies As on LJ

Heterogeneous, coarsely granular suspension

As on LJ

Heterogeneous, coarsely granular suspension

Uniformly homogeneous yellow suspension Uniformly homogeneous suspension

Grayish white, smooth to rough; 7HlO agar must be supplemented with lysed sheep erythrocytes, or an X disc must be placed on medium Raised or smooth; centers appear elevated and thickened; when observed microscopically, thinner peripheral portions show stranding bacilli; vary in roughness and are most often intermediate between fully smooth and fully rough but may be completely rough or, rarely, completely smooth; pigmentation characteristics as on LJ Smooth, glistening, grayish white, opaque, domed, circular, OS-l.5 mm in diam, with entire margins or, less often, umbonate, 0.6-2.7 mm in diam, with compact, raised centers and flattened, irregular edges Similar to M kansasii, although rhizoids more commonly seen As on LJ

Usually homogeneous suspension

As on LJ

Heterogeneous, coarsely granular suspension

As on LJ

Heterogeneous, finely granular suspension

Nonpigmented, flat, dry, rough, and corded

Uniformly heterogeneous, coarsely granular suspension Uniformly homogeneous suspension

Small, yellow, with compact centers surrounded by fringe of branching filaments visible on microscopic examination; resemble miniature bird’s nest

Usually heterogeneous, finely granular suspension; some isolates give uniformly homogeneous suspension

Usually homogeneous suspension

Uniformly homogeneous suspension Uniformly yellow and homogeneous

QExtracted from references 50, 52, and 128.

ple, M. tuberculosis coloniesare viewed asbeing Colonial morphology is best evaluated with rough, thin, flat, spreading,and friable (easily translucentagarmediasuchasMiddlebrook 7HlO broken) (110). OrganismssuchasM. xenopi and or cornmealagar containing 3% glycerol. Excess M. fortuitum may showfilamentousextensions. moistureor liquid on the agarsurfaceisdetrimen-

18

CERNOCH

ET AL.

tal to the study of morphology and should not be allowed to accumulate. Although microscopic characterization of colony morphology should not be used for definitive species identification, it can be used to provide information about an isolate’s presumptive identity, the presence of a mixed culture, and the selection of biochemical tests.

CUMITECH

16A

Niacin test

Niacin (nicotinic acid) is producedby mycobacteria and used in the synthesisof NAD and NADP. Most mycobacteria possessan enzyme that converts free niacin to nicotinic acid mononucleotide. Organismsthat lack the enzyme accumulate niacin in the medium.M tuberculosis and somestrains of M. simiae, M. chelonae, and M. marinum cannot convert niacin and therefore Growth rate The growth rate of a mycobacteriumis a critical accumulateit. The best results for niacin tests determination. Becauserapid growers may take follow growth on egg-based media.The amountof >2 weeksto show up on primary isolation, the niacin produced is directly related to the amount growth rate shouldalwaysbe determinedfrom the of growth andthe ageof the colonies.If growth on subculture.Either agar-basedor egg-basedmedia the medium is confluent, the coloniesshouldbe can be used for determining growth rate; the scrapedsothat the mediumwill be exposedto the inoculum should be diluted enough to demon- liquid for extraction. The water-solubleniacin is strate isolatedcolonies.Visible coloniesare pro- extracted from the mediumand then reactedwith ducedin <7 daysby rapid growersand in >7 days chemicalsor tested by a paper strip method (65, by slowgrowers. 139).Resistanceto multiple drugsmay render M. The growth rate is definedasthe growth rate on tuberculosis niacin negative. solidmedia,not in the BACTEC systemor other liquid media. Organismshave shorter detection Nitrate timesin liquid mediathan in solidmedia.A MAC Mycobacteria vary in their abilities to reduce isolate may take 3 to 4 days to grow in the nitrates; this ability is directly related to the culBACTEC systemand 3 to 4 weeks to grow on ture’s age, incubation temperature, and several solidmediabut is still considereda slowgrower. other factors (120). Consequently,there can be problemswith the sensitivity and reproducibility of the nitrate test. Slow growersshouldbe 3 to 4 Photoreactivity Mycobacteria are categorizedinto three groups weeksold before being tested,and rapid growers based on the pigmentation and production of should be 2 weeks old. A variety of methods, including chemical reagents or reagent-impregcarotenoidpigments. nated paper strips,can be used. (i) Photochromogensproduce nonpigmented colonieswhen grown in the dark. However, these Catalase colonies become pigmented after exposure to With the exception of someisoniazid-resistant light. Some mycobacteriarequire extended light strainsof M. tubercdosis, mostmycobacteriaproexposurefor pigmentto be produced. duce catalase.The enzymeis capableof splitting (ii) Scotochromogens produce pigmentedcolohydrogenperoxide into water and oxygen;oxygen nies when grown in light or dark. Some strains bubblesare produced during the reaction. Two may showincreasedproduction of pigment upon types of catalasetests are widely usedto identify exposureto light. All scotochromogens shouldbe mycobacteria.The semiquantitativecatalasetest testedfor photochromogenicityat 25°C in order determinesthe amount of catalaseproduced by to detect1M.szuZg~~, which isa photochromogenat the isolate (69), whereasthe 68°C catalasetest this temperature. detects whether the organismproducesa heat(iii) Nonphotochromogensare nonpigmented stableor a heat-labilecatalase(71). The presence in the light and dark. They may have a paleyellow of Tween 80 in the test solution helpsmaximize pigmentthat doesnot becomemore intensewith the detection of catalaseby dispersingclumpsof exposureto light. mycobacteria.The height of the bubblesseenin Photoreactivity shouldbe tested for on young the semiquantitative test indicates whether the cultures,as it is usually more easily detected on isolateis a low or high producer of catalase(<45 isolatedcolonies.All tubesof mediainoculatedto or >45 mm of bubbles,respectively).If the organdeterminephotoreactivity shouldbe shieldedwith ism has an optimal temperature of 30°C and will aluminumfoil during incubation.When growth is not grow at 37”C, the semiquantitativecatalase first detected,the tube isexposedto light and then test shouldbe performed at the lower temperareturned to its shield. Pigment production may ture. A positive 68°Ccatalasetest is indicatedby take longer at 25 than at 37°C.Interpretations of the formation of bubbles,howeverfew. The tubes pigmentation patterns must be carefully evalu- should be handled carefully to avoid forming ated, since variations within speciesoccur (98). bubbles from Tween 80 which could be interFor example, some MAC isolatescan be pig- preted incorrectly as a positive reaction (false positive). mented.

CUMITECH

16A

Tween80 hydrolysis Tween 80 hydrolysismedium contains the detergentTween80 (polyoxyethylenesorbitanmononucleate)and neutral red. When the neutral red is bound by the Tween 80, the mediumis an amber to straw color (132). Lipasesproduced by the mycobacteriasplit the Tween 80 into oleic acid andpolyoxyethylated sorbitol. Sincethe Tween80 is no longer intact, it no longer binds the neutral red, and the mediumis red to pink at pH 7. A concentratedsolution of the Tween 80 hydrolysis substrateis commerciallyavailable, producesexcellent results, and is extremely easy to make. Caution shouldbe usedwhen interpreting Tween 80 results;the test shouldbe consideredpositive only when the medium turns red to pink. Cells that are red or pink do not qualify as a positive test. Arylsulfatase Arylsulfatase is an enzyme that hydrolyzes bondsbetweenthe sulfategroup and the aromatic ring of tripotassiumphenolphthaleinto form free phenolphthaleinand remaining salts (128). The free phenolphthalein is detected by adding an alkalinereagent(sodiumcarbonate)to produce a red diazo reaction.A 3-day test helpsidentify the potentially pathogenicM fortuitum group and the 1M.chelonaegroup, while a 14.day test aids in identification of M. ma&urn, M. szulgai, M. xenopi, M. smegmatis, and M. triviale (136). Two methodsare availablefor the test: the Wayneagar testfor the 3-daytest, and the broth of Kubica and Rigdon (72) for both the 3-day and the 14-day tests.Two different concentrationsof tripotassium phenolphthaleinsulfate are needed:0.001M for the 3-day test and 0.0003M for the 14-daytest.

DIAGNOSIS

OF THE MYCOBACTERIOSES

19

ism and most isolatesof M. chelonae) and M. triviale are salt tolerant (106). M. flavescens has a varying tolerance to NaCl and may or may not grow. Iron uptake Iron is neededby bacteria for the synthesisof proteins. When the mycobacterial cell takes up iron from the medium, both the colony and the mediumchangecolor. The iron uptake test determines the ability of an isolate to convert ferric ammoniumcitrate to iron oxide. M. chelonae and M. abscessus are negativefor the iron uptake test, while the M. fortuitum group andM. smegmatis are positive. The conversionto iron oxide resultsin a rusty brown color in the coloniesand medium. Isolatesof the M. chelonae-likeorganismproduce a tan color most noticeable at the colony edges (106). Urease The ureasetest is a simplemethod for distinguishingamongM. scrojdaceum, M. szulgai, and M. gordonae. Urease hydrolyzes urea, forming ammoniaand changingthe pH of the medium;the resulting alkaline pH changesthe phenol red indicator to pink (positive reaction). Three different methodsare available: the Murphy-Hawkins disk method (82), the Steadhammethod (ill), and the Wayne method (130). All three are commercially available and easy to perform. If the Steadhammethod is used,a non-CO, incubator must be usedto avoid false-negativeresults.

Growth on MacConkey agar without crystal violet Growth on MacConkey agar without crystal violet can help distinguishamongthe rapid growTellurite reduction ers. The M. fortuitum group and the M. chelonae Potassiumtellurite is reducedto black metallic group both grow on MacConkey agar and may tellurium in 3 days by MAC strains (61). Some sometimesproducea color changein the medium rapidly growingmycobacteriaalsoreducetellurite in 5 to 10 days. Except for some strains of M. in 3 days.The 0.2% potassiumtellurite is addedto smegmatis, saprophyticmycobacteriado not usuan actively growingculture of MAC in 7H9 broth ally grow in this medium. with Tween and examinedafter 3 days.A black precipitate indicatesreduction to tellurite. Temperature studies The growth rate of a particular isolateat various Susceptibility to TCH temperatures(24,32,37, and42°C)canbe helpful Since some strains of M. bovis may produce in preliminary identification and the selectionof small amountsof niacin and slightly reduce ni- subsequentbiochemicaltests. If slow growth octrate, they maybe difficult to differentiate from M. cursonly at 37 and not at 24 or 42”C,the isolateis tuberculosis. M. bovis is susceptibleto TCH at 5 likely to be from the M. tuberculosis complex, pg/ml and therefore will not grow on the medium, which includesM. bovis. Slow growersat 37 and whereasM. tuberculosis is resistantand will show 42°C with no growth at 24°C may be from the growth. MAC (109). Temperature studiesshould always be performed on isolateswhen needed. Toleranceto 5% sodiumchloride Few mycobacteriacan grow in the presenceof Utilization of carbon sources Rapidly growing mycobacterialspecies,includ5% NaCl. Pathogenic rapidly growing species (with the exception of the M. chelonae-likeorgan- ing major pathogensand infrequent isolatessuch

20

CERNOCH

ET AL.

CUMITECH

as M smegmatis and the M. chelonae-likeorganism,canbe differentiatedby their abilitiesto grow on minimal medium containing sodium citrate, mannitol, or inositol as a sole sourceof carbon (106, 122, 124). Susceptibility

to polymyxin

B and amikacin

Identification of the M. fortuitum complexto the specieslevel is becomingincreasinglyimportant becauseof the differencesin the antimicrobial susceptibilitiesand clinical diseasepresentations of M. fortuitum and M. chelonae. The disk diffusion method may be used with polymyxin B to separatethesegroups.Any zone of complete or partial inhibition by polymyxin B identifies the organismas part of the M. fortuitum group, and the lack of a zone of inhibition identifies the organismasM. abscessus or M. chelonae (125). The amikacindisk can be usedto confirm this identification, with the M. fortuitum group generally showingzone sizesof 230 mm andM. chelonae and M. abscessus showingzone sizesof ~30 mm. Pyrazinamidase

The enzyme pyrazinamidasehydrolyzespyrazinamide to pyrazinoic acid and ammonia; the pyrazinoic acid is detected by the addition of ferrous ammoniumsulfate to the medium (130). A positive test is indicated by a pink ferrouspyrazinoic acid complex.The test is mostusefulin differentiating M. bovis from M. tuberculosis and M. marinum from M. kansasii. NAP

NAP is an intermediate in the synthesisof chloramphenicol.Mycobacteria belongingto the M. tuberculosis complex(M. tuberculosis, M. bovis, andM. aJiiCanum) are inhibited by the presenceof NAP, while NTM are inhibited only slightly or not at all. To determinethe inhibition of growth, the production of 14C02in a BACTEC vial is monitored. A decreaseor slight increasein the GI of the BACTEC vial along with an increase of growth in the BACTEC control vial confirmsthe identification of the M. tuberculosis complex. Using this system,the M. tuberculosis complex and NTM can be differentiatedwithin 5 daysof isolation (84, 98). The manufacturer’sinstructions for the NAP test must be followed. The inoculum should be prepared from actively growing cultures in 12B vials that have reached GIs of 50 to 100, or mycobacterial colonies growing on solid media shouldbe inoculatedto a 12Bvial and allowedto reachthe appropriateGI prior to beingusedasan inoculumin the NAP test vial. Inocula surpassing a GI of 100 are too heavy and must be diluted accordingto the manufacturer’sinstructions(98). Immediateinoculation of the NAP test vial with

16A

organismsharvesteddirectly from solid medium to a 12B vial or directly from smear-positive specimensshouldbe avoided. Results of the NAP test must also be interpreted carefully and in conjunction with the isolate’smorphologicand growth characteristics.Although membersof the M. tuberculosis complex are inhibited by the 5 mg of NAP impregnated into a paper disk found in the vial, they may still showslightincreasesin the GI during the first few days of incubation. Therefore, a trend shouldbe obtained to showthe necessarysignificantmetabolic decreases on two consecutivedays.M. tuberculosis complex should not be reported if the positive control does not grow within the prescribedperiod (approximately5 days).Someother mycobacteria(e.g., M. kansasii, M. gastri, M. szulgai, M. terrae, and M. triviale) may require additional incubation time (becauseof slowergrowth or longer lag times) to showtrends of increasing GIs. M. kansasii may be inhibited by the BACTEC PANTA treatment, thereby allowing minimal growth in the NAP test and potential false-positive results.Attempts to perform the test at nonoptimal growth temperaturesfor the isolatebeing studied (e.g., M. marinum, M. chelonae, and M. xenopi) may result in erroneousreadings;control vials shouldbe observedat all times.A significant increaseor decreasein the GI is defined by the manufacturerasa changeof at least20% between two consecutivereadings.If differencesare borderline or just above 20%, the test should be repeatedthe following dayand carefully evaluated for a trend if resultsare still within closeparameters. Mixed infectionswith M. tuberculosis and NTM may occur; in these cases,the presenceof the NTM will provide an increasingGI, thereby masking the presenceof M. tuberculosis. Culturescontaminatedwith bacteria can alsoshowincreasing GIs. NAP test resultsshouldalwaysbe confirmed by either conventionalbiochemicalor DNA probe studies.The recent introduction of simple,more cost-effective nonradiometric probe technology for identification of mycobacteria from culture may obviate the useof the NAP test in the future. DNA probes

The newest technology for the mycobacteria laboratory involves DNA probes, which allow rapid identification (in approximately 2 h) of the most commonly encountered mycobacteria and eliminate the necessityof numerousbiochemical testsfor the identification process.However, poor sensitivityand specificityprecludethe useof these probesfor direct mycobacterialdetection in specimensat this time (39, 84). Introduced commerciallyin 1987, the original DNA probes for M. tuberculosis complex, M. avium, M. intracellulare, and M. gordonae were

CUMITECH

16A

DIAGNOSIS

OF THE MYCOBACTERIOSES

21

isotopic (Gen-Probe, Inc., San Diego, Calif.). types have been published (9, 34, 37, 56). In These probes were labeled with 12? and were addition, these methods have been applied to targeted to the more abundant mycobacterial other, more frequently encounteredMycobacterRNA. Although expensive,cumbersometo use, rium spp. (55, 118, 119). and short-lived (half-life of about 1 month), the However, as of October 1994, none of these probesworked with adequatesensitivityand spec- methodshasbeen cleared by the U.S. Food and ificity for identification of mycobacteriaisolated Drug Administration for commercialuseas an in from both agar and liquid culture media suchas vitro diagnosticproduct. Thus, laboratoriesdesirBACTEC 12B and 13A (25, 36, 39, 41, 60, 85). ing to usetheseresearchmethodsare cautionedto Conversionfrom isotopicto nonisotopicnucleic use their own quality assurancevalidation proacid probes having sensitivitiesand specificities gram and clinical evaluations to determine the similarto their original counterpartshasprovided a more practical approach to probe technology performancecharacteristicsof any of thesemeth(53,76,96). Presently,nonisotopicsingle-stranded odsbefore anyattempt to usethem for testingand reporting patient results.A proposedguidelineon DNA probesfor the identification of M. tubercuthe proper developmentandqualification of these losis complex,MAC, M kansasii, andM gordonae are available from Gen-Probe (AccuProbe sys- testsis availablefrom the National Committeefor Clinical Laboratory Standards(87). tem). In attempting to apply one of theseamplificaThe nonisotopic nucleic acid probe test is a homogeneousassaythat uses the hybridization tion methodsto the detection of Mycobacterium protection assayprinciple (4). The test usesacri- spp. in patient specimens,the following general dinium ester-labeled chemiluminescent DNA considerationsshouldbe evaluated.First, the colprobes complementaryto rRNA. After a stable lection, transport, and processingof specimens DNA-RNA hybrid is formed, the acridiniumester must be evaluatedwith respectto eliminatingthe label within the hybrid is protected from hydroly- effects of nucleases,inhibitors, and interfering sis(by a selectionreagent) to a nonchemilumines- substancesand therefore allowing adequate recent form. Thus,when an alkaline(NaOH) hydro- covery of target nucleic acids from infected pagen peroxide solution is added to initiate the tients. Second, methods or mechanismsfor circhemiluminescence, only the hybrid bound acri- cumventing both specimencross-contamination dinium ester is able to emit visible electromag- and carryover amplification contamination must netic energy. The amount of light produced is proportional to the amount of hybridized probe be employed.These effectsneed to be evaluated and is measuredon a chemiluminometer.The by using the appropriate positive and negative reader determineswhether a specimensampleis controls as well as internal controls. Third, the needto be evaluated positive or negative by comparing the sample’s primer and probe sequences light emissionvaluesto thoseof referencecutoffs. for specificity of reaction to a significantnumber The facilitation of use of DNA probes, their of closely related species,genera,and other mianticipatedto be encounteredin the longer shelf life of at least 6 months, and their croorganisms overall accuracy in identification promote them typical specimento be tested. Fourth, the level of for widespreaduse (98). Presently, their limita- sensitivity in the number of organismsthat can be tions include cost of the product, availability of detected from a given specimenvolume must be only a small number of distinct probes, and a determined, with reproducibility at or near the requirement for large inocula. Overall, DNA limits of detection. Finally, the entire assaysysprobesshow >99% sensitivity and specificity for tem, from procurementof specimento interpreM tuberculosis complex isolates and approxi- tation of results,needsto be rigorously evaluated mately 95% sensitivity and >99% specificity for against high-quality culture and identification MAC isolates.Although rare, someinstancesof tests by using a significant number of patient cross-reactions between the M tuberculosis com- specimens representativeof thoseroutinely examplex probe and iW. tewae and M celatum isolates ined in the laboratory setting. The limitations of have beenreported (17, 103). the test systemshouldbe clearly stated,indicating the types of specimensand processingthat are Nucleic acid amplification In recent years, a number of different nucleic acceptableand the need for repeat testingwhen acid amplification schemeshave been developed resultsare not clearly interpretable.As the system to overcomethe limits of sensitivity of standard is standardized,if the problem of contamination DNA probe hybridization detection methods. with endogenousinhibitors is resolved,if the cost Most notableof theseschemes hasbeenPCR (94, does not remain prohibitive, and if additional 101). Resultsof a number of studiesthat used probes are introduced, PCR will become more PCR and other methodsof amplificationto detect accepted and more widely used in the clinical M tuberculosis in sputum and other specimen laboratory.

22

CERNOCH ET AL. TABLE

CUMITECH

9. Antimicrobial

16~4

agents routinely tested against mycobacteria

Organism

Antimicrobial

agent tested

M. tuberculosis . .. . .. . .. . .. . .. . .. . .. . .. . .. .. . .. . .. . .. . .. . .. Isoniazid, M. marinum ,............................................... Rifampin,

rifampin, ethambutol, streptomycin, pyrazinamide ethambutol, trimethoprim-sulfamethoxazole, doxycycline or minocycline, clarithromycin Rapidly growing mycobacteria .................Sulfamethoxazole or trimethoprim-sulfamethoxazole, ciprofloxacin or ofloxacin, amikacin, imipenem, cefoxitin, clarithromycin, doxycycline or minocycline M. hnsasii . . .. . .. .. . .. . .. . .. . .. . .. . .. . .. .. . .. . .. . .. . .. . .. .. . Rifampin, ethambutol, isoniazid MAC ............................................................Testing not currently recommended using standard proportion method concentrations (121)

Chromatographicanalysis SincespecificDNA probespresently have limited availability and remain comparatively costly, another method is neededto provide rapid, inexpensive identifications. Chromatography is a promising tool. Specific methods include thinlayer chromatography(8), gas-liquidchromatography (116), and, especially,HPLC (13-16, 115). The most successfullyapplied methods include the last two. Gas-liquid chromatography and its commercially available modification (Microbial Identification System;Microbial ID, Inc., Newark, Del.) have been usedin severallaboratorieswith someaccuracyfor severalyears (98). The HPLC method,usinganalysisof mycolic acidsasbromophenacyl esters,can provide distinct patterns for individual speciesand isbeingutilized at the CDC and severalstate health laboratories(14, 16, 74, 115). Thesemethodsare rapid (with turnaround timesof a few hours) and comparativelyinexpensive after start-up costsfor chromatographicand computer equipment. Chromatographicpatterns for type strains of eachspeciescanbe storedin computermemoryto match unknownisolatesto the known type strain. One disadvantageof thesetests is that they can only be performed sequentiallyin the chromatographic columnsand cannot be batchedfor sample testing. Also, these methodsrequire a dedicated, skilled technologist to maintain the chromatographiccolumnsand instruments. Recently, applicationsof DNA-DNA hybridization techniquesfor speciesrelatednessand HPLC for mycolic acid patternshave facilitated differentiation of species(84,98). Resultsof someof these studieshavereaffirmedor castdoubt on the status of somemycobacterial species.Thus, the separate-speciesstatusesof M avium and M intracelMare were reaffirmed,while that of M aficanum wasdeemedprobably unjustified.

promiseof becomingpractical alternativesfor the rapid identification of mycobacteria in cultures (84, 98, 131). Esoteric identification methodssuch as bacteriophage typing and serotypingmay at times be useful for epidemiologicalstudy (84). However, their general scarcity, poor reproducibility, and poor specificity together with the difficulty of standardizing the test systemshave made them impractical for routine application to mycobacterial identification. SUSCEPTIBILITY TESTING

Antimycobacterial Chemotherapy Five drugs are currently consideredfirst-line mycobacterial drugs, and some combination of theseagentsisrecommendedfor initial therapy of tuberculosisand someNTM diseases. The drugs are isoniazid, rifampin, ethambutol, pyrazinamide, and streptomycin. For patients with pulmonary tuberculosis,shorter coursesof chemotherapy havenow replacedthe traditional 18to 24 months of treatment. Both CDC and ATS currently recommenda 6-monthregimenof isoniazid and rifampin for non-HIV-infected patients (3) and a g-month regimenfor HIV-infected persons (2). Pyrazinamide is also given for the first 2 months. For patients at risk of primary drug resistance,ethambutoland streptomycinare often added until results of drug susceptibility testing are available. CDC recommendsthat initial isolatesof A4 tuberculosis from all patientsbe tested for susceptibility to the primary antituberculous drugs and that susceptibilitytests be repeated if sputumspecimenscontinue to be culture positive after 3 monthsof therapy (Table 9) (114). The ATS recommendsisoniazid,rifampin, and ethambutol for 18 months for patients with 1M. ikansasii pulmonary infections, while isoniazid,rifampin, and ethambutolfor 18 to 24 monthswith streptomycin for the first 3 to 6 months are Other methods Immunologicprobes,usingradioimmunoassay, recommendedfor pulmonary diseasewith MAC enzyme-linkedimmunosorbentassay,and immu- (123).Rifampin plusethambutolfor 3 to 6 months noblot technology,have alsobeen developedre- is sometimesusedfor initial therapy for patients cently. Thesesystems,directed againstfairly crude with Ml marinum. For patients whose isolate preparations of mycobacterial antigens, show becomesresistant to these drugs or for MAC

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infectionsthat fail to respondto primary therapy, a secondgroup of sevendrugs,consideredsecondline agents,may be used.Thesedrugsinclude the injectable agents kanamycin (or amikacin) and capreomycinand the oral agentspara-aminosalicylic acid (PAS), cycloserine,ethionamide, and ofloxacin (or ciprofloxacin). In general, these agentsare more toxic and lesseffective than the first-line agents(121). A numberof additional oral agentshave shown promise for therapy of disseminatedMAC disease.Clofazimine,a traditional drug for the treatment of leprosy, hasfrequently been usedin the treatment of AIDS patients with disseminated MAC disease.Rifabutin (Mycobutin; formerly ansamycinor LM-427) hasrecently beenshownto be highly effective in preventing disseminated MAC diseasein AIDS patients with low CD4 counts (44). Recommendationson how this drug shouldbe usedin treating establisheddiseaseare not yet available. The newer macrolidesazithromycin and clarithromycin have shown excellent activity in monotherapyclinical trials againstdisseminatedMAC disease(30) and are considered more active than any of the traditional antituberculousagents. A numberof traditional antibacterialdrugsare now routinely usedin the therapy of NTM disease due to M. marinum and the rapidly growing mycobacteria.Minocycline, doxycycline, and trimethoprim-sulfamethoxazoleare often used as single-drugtherapy for M marinum asan alternative to rifampin plus ethambutol (121, 137). For the commonpathogenicrapidly growingmycobacterial species(M fortuitum, M abscessus, and 1M. chelonae), the first-line antituberculousdrugsare ineffective in vivo and in vitro. Therapy includes injectable agents(e.g., amikacin, imipenem,and cefoxitin) and oral agents (e.g., sulfonamides, doxycycline, and the newer fluorinated quinolones) (112, 113, 126). Because of speciesand subspeciesvariations in susceptibility to these agents,therapy with these antimicrobial agents shouldbe basedon in vitro susceptibilities.The newermacrolides,especiallyclarithromycin, have shownexcellent in vitro activity against100% of isolates of M abscessus and M chelonae and approximately 80% of isolates of M fortuitum (11) . .. Traditional susceptibilitytesting and radiometric susceptibilitytesting of M tuberculosis and NTM are describedin detail elsewhere(49). However, owing to the increasingincidenceof clinical diseasedue to rapidly growingmycobacteria,agar disk elution and broth microdilution methodsfor theseorganismsare describedbelow. Agar Disk Elution Methods The agar disk elution method for M marinum or the rapidly growing mycobacteriais similar to

DIAGNOSIS

OF THE MYCOBACTERIOSES

23

that describedfor M tuberculosis but with some modifications.Because7HlO agar (and its pH of 6.6 to 6.8) hasa significanteffect on the activities of a number of antibiotics, susceptibility tests should utilize Mueller-Hinton agar, which has a pH of 7.4. OADC (oleic acid, albumin, dextrose, and catalase) enrichment is added to a final concentrationof 10%to enhancethe growth of 1M. chelonae, M. abscessus, andM. marinum. Because of problemswith adequatedrug diffusion into the corners of the traditional quadrant plates (lo), round-well tissue culture plates that contain six round wells, each 3.5 by 1.0 cm (Becton Dickinson), are preferred. For plate preparation,commercialsusceptibility disks are placed in the center of the wells and covered with 0.5 ml of OADC for at least 60 s. Then, 4.5 ml of molten Mueller-Hinton agar is added.The contentsof the platesare swirledwith a sterile stick (the wooden end of a sterile swab alsodoesfine) to provide goodmixing of the drug, the disk(s)is centered,and the mediumis allowed to harden. Growth control wellswithout drug are included for each organismdilution. The plates can be used immediately, but absorption of the inoculum may be slow if some amount of water evaporation is not allowed. Becauseof the instability of the tetracyclines and imipenem,plates shouldbe refrigerated and not left out overnight to dry. The stability of the tetracyclines and imipenemwhen refrigerated is variable; hence, plates should be used within 3 days of preparation. Becauseof this instability, platesto be usedfor specimentesting shouldnot be incubatedovernight asa sterility check. For inoculation, dilutions of the organismare preparedin broth or in distilledwater aswith 1M. tuberculosis. In general,a suspension of organisms that matchesthe 0.5 McFarland barium sulfate standardis madeand then diluted 1:lO and 1:lOO. A lo+1 sampleis addedto the surfaceof the agar, which is tilted to allow somespreadof the inoculum. Larger volumesof inoculumare discouraged only becausethey are absorbedpoorly, given the usualhigh degreeof hydration of the plates.The platesare coveredand then placedin a 28 to 32°C incubator. The addition of 5 to 10% CO2 hasno known benefit for the growth of M fortuitum, M chelonae, M. abscessus, or M. marinum. The useof a highly moisturizedincubator may inhibit absorption of the inoculumif the water vapor pressureis too high. Susceptibilityplatesfor the rapid growers are incubated for 3 days, with an additional 1 to 3 days required for some isolates of 1M. abscessus and most isolatesof M. chelonae. Susceptibility platesfor M marinum are incubatedfor 7 days. The inoculumconcentrationchosenfor reading is the one that resultsin 100to 300CFU. Susceptibility is read as complete inhibition of visible

24

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growth on the drug plate compared to the growth control, except for sulfisoxazole or trimethoprimsulfamethoxazole, for which ~80% inhibition of colony size is used as the endpoint. The endpoint for this method is usually sharp (no growth or growth equivalent to that of the growth control). Problems-with this method include the inability to test erythromycin and the absence of large-scale comparisons of the results of this method with those of other methods. These problems and other details of this method are well reviewed elsewhere (11, 49). Broth Microdilution Method A broth microdilution method for susceptibility testing of rapidly growing mycobacteria was first described by Swenson et al. (112). It utilizes cation-adjusted Mueller-Hinton broth. Some of the drugs recommended for testing against rapidly growing mycobacteria are available in commercial broth microdilution panels routinely used for testing gram-positive or gram-negative bacteria, while some drugs (e.g., doxycycline, minocycline) and some drug concentrations (such as those used for cefoxitin or ceftmetazole) may not be available in those plates. Organisms are prepared from a broth culture or by scraping them from an agar plate and suspending them in broth. The use of sterile glass beads (0.5 mm) or 0.02% Tween 80 helps disperse the organisms. The organism suspension is prepared to the optical density of a 0.5 McFarland standard (approximately lo7 to lo8 CFU/ml). The suspension is then diluted to produce a final inoculum in the well of lo4 to lo5 CFU/ml. The plates are sealed, placed in plastic bags, and then incubated at 28 to 30°C in a humidified ambient-air environment. The plates are incubated for 3 days, although some isolates of iV. abscessus andmanyisolatesof 1M.chelonae require an additional 1 to 3 daysof incubation (a total of 4 to 6 days)to producesatisfactorygrowth. The MIC is read as the lowest concentration that produces no growth observable with the naked eye except on plates containing sulfonamides or trimethoprim-sulfamethoxazole,for which ~80% inhibition of growth is the endpoint. This endpoint is generallysharpfor all the drugs used.MIC breakpointsfor rapidly growing AFB in broth microdilution testsare the sameasthose for aerobicbacterialisolates,with the exceptionof that of cefoxitin, for which a MIC of >32 rather than X6 pg/rnl (113) is consideredto indicate resistance.Quality control with this method can be done by using the National Committee for ClinicalLaboratory Standards-recommended bacterial strains, P. aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 29213. The type strain of M fortuitum (ATCC 6841) can also be used as a control strain and has been recom-

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mended for this method (113). Acceptable control ranges for this strain are detailed in the fifth edition of the Manual of Clinical Microbiology (49) . COMMONLY ISOLATED MYCOBACTERIA The mycobacteria are a diverse group of AFB with high lipid content. The lipids in their cell walls include waxes with characteristic long-chain (60- to 90-carbon) mycolic (fatty) acids. The mycobacteria are normally straight or slightly curved rods measuring 0.3 to 0.6 Frn by 1 to 4 km. Mycelia are rarely observed; when produced, they are usually rudimentary and fragment easily into coccobacillaryunits. Diversity in the genusis manifestedby variability in morphology, growth rate, nutritional and temperature requirements, and pathogenic@. More than 50 speciesof mycobacteriaare presently recognized,with only approximately 25 speciesbeingcommonlyassociated with humaninfections (43, 84, 135). M tuberculosis, M bovis, M. aficanum, and M. leprae are alwayspathogenic. With the exception of M. leprae, they all cause classic tuberculosis and together form the M. tuberculosis complex.This group alsoincludesM. microti, which is found in certain rodents but not in humans. AL tuberculosis

The most important mycobacterium from a public health perspectiveis M. tuberculosis. It is one of the most virulent of the AFB, and tuberculosis is a highly contagious disease.Public health departmentsmustbe contactedsothat they can deal with contactsquickly. Infection control personnel need the same prompt notification. Serpentine-cordformation, producedby a glycolipid (trehalose-6,6’-dimycolate,or cord factor), occursin liquid mediaand is mostpronouncedin media lacking a wetting agent. This phenomenon is also noted in BACTEC media and aids in preliminary identification. Colonieson LJ usually appearin lessthan 3 weeksandhave a rough, buff, warty, granular or cauliflower morphology.Drugresistantorganismsmay take 4 to 6 weeksto grow. The coloniesare easily picked from solid media but are hard to emulsify.Therefore,vortexing with glassbeadsis often neededto help dispersethe clumps of organisms.Dry, rough microcolonies may appear on 7Hll in lessthan 1 week but are much flatter than they would be on egg-based media. Recently, the CDC publishedthe secondedition of draft guidelinesfor preventing the transmissionof tuberculosisin health care facilities (20). These guidelinesinclude recommendations for laboratoriesthat evaluate patients for tuberculosis.In order to isolatepatientswho may have multidrug-resistantM. tuberculosis assoonaspos-

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16A

sible, the CDC recommendsthat resultsof AFB smearsof sputumbe available in lessthan 24 h after specimencollection;that detection and identification of 1M.tuberculosis be completedwithin 14 and 21 days of specimencollection, respectively; andthat susceptibilitytestingof 1M.tuberculosisbe completedwithin 28 days of specimencollection (19 . M. bovis

Infection with IM. bovis is uncommon in the United States, but the organismis a significant pathogenin other parts of the world. One of the major reasonsfor the decreasein the number of infectionsis the pasteurizationof milk. However, strainsof IM bovisfrom the BCG vaccine,which is usedto treat malignantmelanomasand carcinoma of the bladder, can be isolatedfrom inoculation abscesses, regionallymph nodes,and urine. The growth of 1M.bovis is not stimulated by addedglycerol and may in fact be inhibited by it; conversely, LJ with 0.4% pyruvate enhances growth. Becauseof the lower glycerol content of Petragnani medium, it may be preferred over other egg-basedmedia.1M.boviscoloniesare buff, low, and small; emulsify easily in solution; and usuallyappearwithin 3 to 6 weeksin culture. The colonies are sometimesdescribed as pyramid shaped.When agar-basedmedia are used, the coloniesresemblethoseof M tuberculosis but can be differentiated from them by negative results from a nitrate test, resistanceto pyrazinamidase, and sensitivity to TCH. M. a@canum africanurn is consideredintermediate between M. tuberculosis and M. bovis, but it more closelyresembles M. bovis. Whether this organism is a distinct speciesor a subspecies or biovar of M. bovis is still undecided,sincebiochemicalresults for M. africanurn isolatesthemselvesare variable M

(134). The organism is biochemically inactive, with the only reactive test being a positive urease test.It alsoexhibitsvariable sensitivityto TCH. M. aficanum is rarely isolatedin the United States; mostisolatesoriginate in Africa. M. ulcerans M. ulcerans is a slowly growing nonpigmented organismisolatedfrom skin ulcers (Buruli ulcer) in the tropics. The organismhasnot beenseenin the United States, but casescan be found in Australia, Malaysia,Africa, and Central America. Isolation of the organismmay take 6 to 12weeks at 30 to 33”C, and it doesnot grow at 37°C.M. ulcerans is not isolatedfrom sputum. M. marinum M. marinum was first found as a pathogen of fish (5) and is now found in chronic ulcerating

OF THE MYCOBACTERIOSES

25

granulomasof the skin that usually result from trauma to skin before or during water exposure. The diseaseis commonly referred to as “swimmingpool granuloma”andmay be associatedwith fresh or salt water, aquariums, and industrial exposure.The organismgrowsoptimally at 30 to 33°C; at 37°C it may not grow at all or may grow only upon repeated subculture. This photochromogenis not usually found in sputum. M. haemophilum

One of the mostrecently describedmycobacteria, M. haemophilum, hasbeen isolatedfrom skin lesions, synovial fluid, and bone marrow from lymphoma and renal transplant patients. More recently, most isolateshave been from patients with AIDS, although M. haemophilum has also been isolatedfrom cervical lymph nodesin nonimmunocompromised children. Like M. marinum andM. ulcerans, its optimal growth temperatureis between30 and 33°C.The organismhasan absolute requirement for hemin and may be isolated on chocolate agar, LJ with 1% ferric ammonium citrate, or Middlebrook agarenrichedwith hemin. Coloniesare smoothto rough and nonphotochromogenic.A simplealternative to usingthe specialized LJ or Middlebrook media for growth of the organismis using X-factor strips (Difco, BBL, Remel) on Middlebrook 7HlO plates. M. haemophilum growsaroundthe X strip within 2 weeks at 30°C while there is no growth on a plate without the strip (117).The BACTEC systemmay be used to grow the organism by adding 5% hemolyzedwhole humanblood (28) to a 13A vial. M. kansasii

The most commonly isolated photochromogenit mycobacteriumisM. kansasii. The organismis usually isolated from pulmonary specimens,but isolationfrom bonemarrow,joint fluids, skin, and lymph nodes has been reported. Dissemination can occur in patients with someimmunological deficiency. Middle-aged white males have the highestincidenceof chronic lung disease,but all agegroupscan be infected (1). Colonization and contaminationcan occurwith this organism,but in patients with underlying pulmonary disease,the risk of infection is high (75). Microscopically, the organism appears long, broad, andbeadedor banded.On solidmedia,the photochromogeniccoloniesusuallyappearafter 2 weeksof incubation. Rare isolatesof M. kansasii have been reported to be scotochromogensand nonphotochromogens. Fully rough to fully smooth colonies may appear along with intermediate forms. Isolation in the BACTEC systemusually occursin lessthan 10days.An acid-faststainfrom the BACTEC bottle may showloose cording, so closeexamination of the smearalong with comparisonof GIs and recovery time is necessaryto

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make a presumptive differentiation from IM. tuberculosis. M. kansasii

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at 37°C and is absentat 42OC.This organismmay be confusedwith M. flavescens, but the photoreactivity of the former at 25 but not at 37°Cshould assistin distinguishingbetween them. M. flavescens usually growsin the presenceof 5% NaCl, whereasM. szulgai doesnot. Of the scotochromogens,M. szulgai is usually the most sensitiveto ethionamide,rifampin, ethambutol,and high levelsof isoniazid.

strains are strongly nitrate and ureasepositive and are Tween 80 hydrolysispositive in lessthan 5 days(many timeswithin 24 h). Most strainsdo not grow at 42°C.Strainswith high catalaseactivities are usually considered more virulent than low-catalaseproducers. Resistance to PAS is normal, and susceptibilityto isoniazid and streptomycinvaries.All pretreatment isolates shouldbe susceptibleto rifampin. M. gordonae If the NAP test is usedfor identification of M. The scotochromogenM. gordonae is usually tuberculosis, it shouldbe interpreted with caution, isolatedasa singleyellow-orangecolony or a few becauseM. hnsasii may alsoat timesbe inhibited isolatedyellow-orangecoloniesafter 3 weeksor by NAP. more at 37°C. It is commonly isolated from tap water and will colonize water taps and stills. If a M. simiae laboratory is observingfalse-positiveAFB smears, The first isolatesof M. simiae were from mon- the water sourceshouldbe checkedfor M. gordokeys.The associationof diseasewith monkeysand nae. Rubber hoseson water spouts should be monkey caregivershas led to somespeculation replaced regularly, becausethey also harbor the about the zoonotic epidemiologyof this disease. organism.It is most likely to be mistakenfor M. The organismscan alsobe isolatedfrom environ- scrofulaceum but is distinguishableby a positive mental sourcessuchastap water (137). Tween 80 hydrolysis test and a negative urease Smooth, moist colonies usually appear in 2 test. It is usually not a recognizedhuman pathoweeksat 37”C,and the morphologycan be similar gen. to that seenwith MAC, i.e., domedcolonies.The organismis most commonly photochromogenic, M. scrofiluceum but the photoreactivity is unstableand often deM. scrofilaceum is a commoncauseof cervical layed. Eight hours of light exposure (24 h is necessaryin rare cases)is neededto induce the lymphadenitis(scrofula) in children. Primary dislight reaction. Growth isusuallypoor at 25”C,and easein adultsis uncommon,but the organismmay there is no growth at 45°C. The niacin test has colonize old tuberculouscavities.The organismis been reported to be positive but lacks reproduc- common in the environment, so careful assessibility (133). Becauseof the niacin results, this ment of pathogenicity is necessary.Coloniesare smooth,domed,glistening,and scotochromogenic organismmay be confusedwith M. tuberculosis; however, in addition to a positive 68°C catalase when isolated at 37°C in 2 weeks or more; pigand a positive Tween 80 hydrolysistest, the pho- mentation may increasewhen the colonies are toreactivity of M. simiae shouldidentify it. Resis- exposedto light, becomingdark orangeor possitance to a number of antituberculosisdrugs is bly brick red. Sometimesconfusedwith M. gordonae, it can be identified with a negativeTween 80 reported. hydrolysis test and a positive urease test. M. scrofulaceum is often resistant to isoniazid and M. asiaticurn Pulmonary isolatesof M. asiaticum have been PAS. reported, althoughinfectionsin the United States M. xenopi have been rare. The smooth coloniesof the orM. xenopi wasfirst isolatedfrom a South Afriganismare similarin morphology to those of M. simiae but may be differentiatedby negativeniacin can toad and may causepulmonary diseasein and Dveen 80 hydrolysistests.Moreover, a nega- humans.The usual hosts are middle-agedmen tive nitrate reduction test can help separatethis with predisposingpulmonary disease.Hot-water organismfrom M. kansasii. systemsin both the United Statesand Britain have been known to be contaminatedwith this organM. szulgai ism (12,45). The scotochromogenic coloniesgrow M. szulgai has been isolated from the lung, slowlyon LJ, usuallyappearingin 4 weeksor more lymph nodes,and olecranonbursa and from pa- at 37OC.Coloniestypically appearassmallerect or tients with tenosynovitis (32). Smooth or rough tiny domed, brightly pigmented colonies; occacolonies may be isolated after 2 to 4 weeks at sionally,the organismis nonpigmented.When it is 37°C. The organismis scotochromogenicwhen grown on Middlebrook 7H10, short tufts of aerial grown at 37°Cbut photochromogenicwhen grown hyphae may be seen.Branching and filamentous at 25”C, sometimesrequiring 18 h of light expo- extensions,sometimesreferred to as“a miniature sure.Growth of the organismis slowerat 25 than bird’s nest,” may be seenmacroscopicallywith a

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DIAGNOSIS

OF THE MYCOBACTERIOSES

27

temperature varies, with some strains growing best at 42OC. When the BACTEC systemis used for isolaM. jhvescens tion, growth may be detectedextremely rapidly. In M. flavescens is a saprophytemost commonly somecaseswith heavily positive smears,as for grouped with the slowly growing scotochromo- someAIDS patients, BACT.EC bottles may be gens,although it can grow on solid media in 1 positive in 1 to 2 days. Smearsfrom the vials week or less.It can be distinguishedfrom other shouldshowpleomorphicAPB and usuallyexhibit scotochromogens by a positive nitrate reduction a rapid rise in the GI to 999. In addition to this organismcausingdisease,it test and growth on LJ with 5% NaCl. may alsobe a saprophytebecauseof its ubiquitous M. malmoense nature. M. malmoense is associatedwith pulmonary MAIS versusMAIS Intermediate disease.Coloniesusually grow in 4 weeksat 37°C but take much longer (4 to 6 weeks) to grow at The term MAIS refers to Mycobacterium scrofilaceum complex 22OC. The colonies are smooth, glistening, avium-M. intracellulare-M. opaque, and nonpigmentedor grayishwhite. An and includesthose MAC organismsthat are pigunusual property of this organismis a positive mentedassoonasmicrocoloniesappear;pigment Tween 80 hydrolysistest; this test result is usually may rangefrom paleyellow to deeporange.These not seen among pathogenic mycobacteria. It is organismsoften fit the biochemical profile for resistantto isoniazid,PAS, streptomycin,and ri- MAC except for the pigment. Pigmented MAC fampin and susceptibleto ethambutol, cycloser- are difficult to work with, as are thoseorganisms consideredintermediatesbetween MAC and M. ine, and ethionamide. scrojdaceum (MAIS intermediates). The MAIS MAC intermediateshave biochemicalpropertiesof both The MAC includesM. avium and M. intracelluMAC andM. scrofilaceum (48) and do not aggluZare. The organismsare biochemically indistin- tinate with any of the antisera for the MAIS guishableand may be difficult to separateexcept complex.More taxonomicwork needsto be done by molecularmethodsor HPLC analysis.In most to differentiate these organismsand help detercases,however, it is not clinically significant to mine if they are clinically significant. separatethe strains.Pulmonaryisolatesare comM. gad monlyseenin patientswith evidenceof underlying chronicpulmonarydisease.DisseminatedandpulThe nonphotochromogenM. gad, alsocalled monarydiseases are commonin AIDS patientsin the J bacillus,is often isolated asa singlecolony disproportionatenumbers.By usingDNA probes, from gastric aspiratesand is considereda nonDrake et al. determined that 90% of AIDS pa- pathogenicsaprophyte.The coloniesare smooth tients’ isolates are M. avium, whereas 66% of to rough and appear in about 2 to 3 weeks.M. non-AIDS patients’ isolatesare M. intracellulare gastri can easilybe distinguishedfrom MAC by its (36). MAC is difficult to treat, as it is extremely positive ureasetest. drug resistant.New drugs,suchasrifabutin, clarM. genavense ithromycin, and azithromycin, have recently been found to be active against the organism. CurM. genavense is the proposednamefor a newly rently, MAC is responsiblefor more clinical infec- recognized, fastidious, slow-growingmycobactetious diseasescaused by APB than any other rium first isolatedfrom patients with AIDS (7). mycobacterium. The organism is known to cause disseminated Microscopically,the organismisshort and pleo- illnessoften indistinguishablefrom that causedby morphic; branching may be present. Growth on MAC (27). It is now known to be associatedwith solidmediamay take 3 or moreweeks,with better infection of the gastrointestinaltract, liver, spleen, growth seenon Middlebrook agar than on LJ. lymph nodes,bone marrow, and blood. Coloniesare usually smooth,dome shaped,and When studiedby thin-layer chromatography,M. buff; rough and wrinkled coloniesmay also ap- genavense resembles M. simiae and M. malmoense pear. Culturesoften appearmixed, becausemore in its mycolic acid composition.However, it hasa than one colony type may form. The centers of unique 16srRNA sequencethat separatesit from coloniesmay alsocrater after 2 to 3 weeks.Very the other two species. commonly,both opaqueand translucentcolonies The organismgrowsslowlyin liquid mediasuch growon Middlebrook 7HlO agar.The coloniesare asthat in BACTEC 13A or 12Bvials and will not usually not pigmentedbut may yellow with age. grow on solid unsupplementedmedia. MiddleMost isolates from AIDS patients are yellow brook 7Hll agarsupplementedwith mycobactinJ within 2 to 3 weeks.The production of pigment is (2 kg in the agar and 50 pg added to the slant not dependenton light exposure.Optimal growth surface)allowsthe propagationof this isolateon dissectingmicroscope.M. xenopi is usually very responsiveto drug therapy.

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solid media, but extended incubation periods of up to 8 weeks may be necessary to perform either isolation or biochemical studies. Colonies appear dense and creamy or flat and dry. M genavense may be presumptively identified by positive test resultsfor catalase,pyrazinamidase,and urease alongwith fastidiousgrowth characteristics. M. celatum M celatum is a new speciesof mycobacterium

that hasbeenrecognizedasclinically significantin the immunocompromisedhost. Isolates in this group are slowgrowing and similarto but distinct from 1M.xenopi. Analysis of mycolic acid composition and 16s rRNA sequencinghas supported the separate-species status of this organism;the speciesnameM. celatum has been proposed.A few of these M. xenopi-like isolates have had cross-reactionswith the M. tuberculosis complex when tested by genetic probe (Gen-Probe) (18, 103). Although changesin the probe analysis protocol may have now alleviated this rare problem, laboratorians must be aware of potential cross-reactions. Many laboratoriesreleasepositive probe resultsfor M. tuberculosis complex aspresumptivependingconfirmation with a niacin test. M. paratuberculosis M. paratuberculosis has been implicated as an

infectiouscauseof Crohn’sdisease,an inflammatory bowel diseaseseen predominantly in the United States and western Europe (22). The organismis extremelyfastidiousand requiresmedia containing mycobactin J. Incubation periods for isolation of the organismhave rangedfrom 8 weeksto 30 months. The organismis biochemically inactive and has been identified by using various molecular DNA-based detection techniques. M. terrae Complex

The three organismsthat makeup the M. terrae complexare M. terrae, M. triviale, andM. nonchromogenicum. M. terrae is alsoknownasthe “radish” bacillusandhasa circular or irregular morphology with a smoothto granular texture. This nonphotochromogenis differentiated from the other organismsin the complex by a positive nitrate reductiontest and no growth on LJ with 5% NaCl. M. triviale, the only slowly growing nonphotochromogenthat will grow in the presenceof NaCl, is also known as V bacillus. The colonies are rough and may be confusedwith those of M. tuberculosis or the rough forms of M. kansasii. M. nonchromogenicum isthe lastof the M. terrae complex and, like the others, is a rare causeof human disease.It is a recognizedcauseof localized hand infections or tenosynovitis following traumas and is the member of the group commonlv associatedwith clinical disease(97). It is

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distinguishableby a negative nitrate reduction test, no growth in the presenceof NaCl, and its unique HPLC mycolic acid esterpatterns. M. fort&urn

Complex

Many of the clinical characteristicsof the M. group and the M. chelonae group are similar. Both groupscan causelocal abscesses at the injection site or after trauma and surgical wounds.Cornea1infections following a penetrating injury have also been reported (126). These rapid growersare alsoassociatedwith pulmonary disease(usuallyM. abscessus), intravenous-catheter infections,woundinfections(following median sternotomiesor breast augmentation),and renal dialysis. They are consideredthe second most commoncauseof NTM infectionsfollowing MAC (42). Resistanceto antituberculousdrugs is routine; antibiotics such as cefoxitin, sulfonamides, tetracyclines, the fluorinated quinolones, clarithromycin (especially for M. chelonae and M. abscessus), and amikacinare usedfor treatment. The 3-day arylsulfatasetest with positive results and the absenceof pigmentationare indicatorsfor separating these two groups from other less pathogenicrapid growers.These organisms,like MAC, are ubiquitous and can be saprophytesas well aspathogens. Rapid growers may not exhibit characteristic rapid growth (7 daysor less)on primary isolation, especiallyif the specimenwas decontaminated. The harshdecontaminatingprocessmay shockthe organismand causethe growth rate to be exhibited only upon subculture.M. fortuitum, M. abscessus, and M. chelonae may grow on a variety of common bacteriologic media, from blood agar plates to blood culture bottles. One of the most common types of culture medium to support growth of these organismsis the type used for growing fungi. The coloniesoften are dry, wrinkled, and flat. Although they may not stain acid fast when taken from a non-acid-fastmedium,the organismwill utilize the fatty acidsand stainacid fast oncesubculturedto LJ or Middlebrook 7HlO. The M. fortuitum group is more sensitive to antibioticsthan M. chelonae or M. abscessus. Nonphotochromogeniccolonies appear wrinkled or matte within 7 dayson LJ and may becometinted slightly green from absorption of the malachite green. Luxuriant growth usually appearswithin 3 days of subculture. When grown on cornmeal glycerol agar or Middlebrook 7H10, short aerial hyphae may be seenon rough coloniesin 1 to 2 days. No growth occurs at 45°C but appears between 24 and 37°C. Like M. abscessus and M. chelonae, M. fortuitum can grow on MacConkey agar in 5 days without crystal violet, but unlike these two species,it is nitrate positive and iron uptake positive. Two species,M. fortuitum andM. peregrinum, aswell as an unnamed“third group”

fortuitum

CUMITECH

16A

DIAGNOSIS

biovariant of M fortuitum are in the M. fortuitum group (93,98). Thesespeciesor taxonsare differentiatedby growth on sodiumcitrate, inositol, and mannitol carbon sourcemedia. M. chelonae andM. abscessus appearassmooth or matte nonphotochromogeniccolonies on primary isolation; colonies may also be rough and wrinkled. Abundant growth appearson subculture in 3 daysand lacksfilamentousextensionslike M. fort&urn. Thesetwo speciesare differentiated by citrate utilization or by HPLC patterns of mycolic acidesters.The presenceor absenceof growth on mediacontaining5% NaCl is generallyunreliable for differentiating the two species.No growth occursat 45°C but growth doesoccur between24 and 37OC.M. chelonae and M. abscessus may be separatedfrom M. fortuitum on the basisof negative nitrate and iron uptake tests.

OF THE MYCOBACTERIOSES

29

and has a high tolerance to heat (to 52°C). It growsmore slowlythan M. phlei but more quickly than slowly growing mycobacteria.It can be distinguished from M. phlei by its uptake of ferric ammoniumcitrate. QUALITY

ASSURANCE

CONSIDERATIONS

A laboratory must take stepsto ensurethat for every procedure,maximumquality standardsare met for eachoutcomeand resultsare obtainedin an accurate and timely manner. Thus, quality assuranceincludesbut is not limited to quality control, which may test the adequacyof individual test systemsor media.In anylaboratory, all media, reagents,and stainsshouldbe clearly labeledwith their names,receipt dates, expiration dates and openingor usedates.

Procedures and Personnel M. smegmatis Procedure manualsmust be readily available. Human isolates of M. smegmatis are almost alwaysclinically significant(122). M. smegma tis is Proceduresshouldbe written to conform to pro-

a rapidly growing, ubiquitous mycobacterium. Coloniesappearbuff, raised,and smoothto mucoid, and they usually grow in 2 to 4 dayson LJ. Approximately 50% develop a yellow to orange pigment with age (~10 days). Except for their smoothcolony morphology and a negative 3-day arylsulfatasetest, theseorganismsare similar to M. fort&urn. Saprophytic

Rapid Growers

M. chelonae-likeorganismis a nonpigmented, rapidly growingmycobacteriumthat is commonin tap water. It is most commonly recovered from sputum,where it is usually a contaminant. It is generallymore susceptibleto antibacterial agents than the M. fort&urn or M. chelonae groupsand is characterizedby susceptibilityto cephalothin,unlike the other rapidly growing mycobacteria.The M. chelonae-likeorganismusually appears mucoid, and it utilizes mannitol and inositol as carbon sources.Members of this group alsohave a unique HPLC pattern (124). M. phlei producespigmentedcoloniesthat are extremely heat tolerant and resistkilling at 60°C. It also produces the growth factor mycobactin required by someAPB. M. fallax is a rapid grower often confusedwith M. tubercdosis becauseof its cord formation. The organismgrowsrapidly at 30°Cand slowlyat 37OC. It hasbeen reported from pulmonary infections but is also isolatedfrom the environment. It can easily be distinguishedfrom M. tuberculosis by growth rate and by negative niacin and urease tests. M. vaccae is a rapidly growing organismseldomly isolatedin the laboratory and considered nonpathogenic. M. thermoresistible was first isolated from soil

cedural-writing guidelinesrecommendedby the National Committeefor Clinical Laboratory Standards (86). Once established,written procedures should be followed by all personnel, and any changesshouldbe evaluatedand approvedby the laboratory director. The proceduresmust provide specimencollection guidelines,adequately identify and process specimens,and support any reports issuedby the laboratory. Documentation of review of preliminary and final resultsshould be maintained, resultsshouldbe correlated with patient identification and appropriateness of quality control parameters, and all quality assurancerecords shouldbe verified prior to releaseof data. Individual personnelworking with mycobacteria must be properly trained under appropriate supervision and must be involved with documented continuing educationprograms.The volumeof work shouldbe high enoughto ensurethe continuedproficiency of the personnel.Personnel shouldbe assignedto the mycobacteriologysection permanently,or if rotation is necessary,they should remain in the sectionfor at least 3 to 6 months (98). Proficiency

The laboratory and personnelmust participate in an acceptableproficiency-testing survey program,suchasthe one provided by the CAP. These surveysshould challengea laboratory’s capabilities with setsof unknown isolatessimulatingpatient specimens. The challengeorganismsor specimensshouldbe treated asregular material from patients’specimens to besttest the accuracyof the systembeingused.Resultsof suchsurveysshould be sharedwith personneland may be usedaspart of a continuing education program and in fulfill-

30

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ET AL.

ment of state or federal licensure requirements (98) The laboratory should maintain a collection of isolates with documented identities to use as internal challenges for personnel performance appraisals, continuing education programs, and quality control studies. Isolates may be obtained from a number of sources, including state and federal agencies, the American Type Culture Collection, proficiency programs (e.g., CAP), and the Trudeau Mycobacterial Culture Collection (at the National Jewish Hospital and Center for Immunology and Respiratory Medicine, Denver, Colo.) (98, 110). Frequently used cultures may be maintained on LJ slants or in Middlebrook 7H9 broth at room temperature if they are subcultured often (i.e., monthly). For storage periods of up to a year, cultures may be held on LJ slants at 4°C; for longer storage periods, isolates may be rapidly frozen in liquid medium (e.g., skim milk, 7H9 broth, sterile water) and placed at -20 or, preferably, -70°C (58, 98). Cultures can be thawed and refrozen several times before a loss in viability begins to occur. If isolates are frozen in skim milk, a hot wire loop may be used to thaw several loopfuls for subculture. The vial can then be replaced at -70°C with ~99% of the liquid thawing. This procedure will maintain cultures for many years without loss of viability. Specimens and Transportation

Specimensand transport methods should be evaluated.Any deviationsfrom appropriate specimen type, volume, or transport techniqueshould be documentedon the worksheetsand the report. Although sputumspecimens neednot be screened for contamination with upper respiratory tract flora, it is useful to document the presenceof lower respiratory tract secretions (pulmonary macrophages, polymorphonuclearcells,and bronchial epithelial cells). Microscopy

Adequacy of acid-fast-stainingproceduresand microscopic evaluation may be ascertainedby including a slide containing mycobacteriaand a slide containing non-acid-fastorganismsin each run of patient slides.Although positive control slidescanbe madein advancefrom suspensions of M tuberculosis H37Rv and used as needed,it is best to useslidesmadefrom excessconcentrated sputum from patients with positive smearsand cultures. If positive sputum is not readily available, it may be possibleto seednegative sputum specimens with known M tuberculosis (6, 58, 98). 1M.scrofulaceum may also be used as a positive control insteadof M. tuberculosis. Presenceof AFB in negativecontrolsmay indicate contaminatedwater or reagentsusedin pre-

CUMITECH

16A

paring the stains. The water supply should be evaluated and can be periodically monitored for contamination (especiallywith M. gordonae or M. terrae) by filtering 1 liter of water through a 0.22~pm-pore-size filter and culturing the filter on 7HlO or 7Hll agar (58). Positive smearsmay be confirmed by a second reader.Someworkersbelieve that smearspositive by the fluorochromestain shouldbe confirmedby the Ziehl-Neelsenor Kinyoun stain (98). All positive smearsshould be saved for at least 1 to 2 monthsfor possiblereevaluation.Positive smears shouldbe documented,and their prevalence(percentageof all smears)shouldbe monitored (58). Deviations from normal percentagesshould be investigated,asshould any trend of ~2% smearpositive, culture-negativeisolates. Specimen Processing

and Culture

Decontamination and concentration protocols can greatly affect mycobacterial recovery rates. Increase or decreasein the harshnessof the decontaminationprocedure (either in exposure time or in percent NaOH) can be directly reflected by a decreasein the numberof mycobacterial isolatesor an increasein the rate of contamination, respectively.To evaluate the adequacyof the systemsbeing used,the laboratory must continually monitor and documentthe contamination rates of clinical specimens.Contamination rates between 2 and 5% are acceptable,while those below 2% or over 5% suggesteither an overly harshor an overly weak decontaminationprocess, respectively.Contaminationratesof over 5% may alsoreflect incompletedigestionof the specimens prior to culture (58). Someworkers recommendseedingautoclaved sputumwith viable M. tuberculosis and processing the sampleas if it were a clinical specimen(6). Such studies may provide earlier clues to the adequacy or harshnessof the decontamination procedurethan would monitoringisolationtrends. Interpretation

of Results

Resultsof direct smearsand culture must be interpreted carefully. For purposesof patient care, sensitivityandspecificityof laboratory methods are lessimportant than positive and negative predictive values.Thus, althoughusuallygood,the predictive value of a positive direct smearfor AFB dependson diseaseprevalencein the area. It is alsofrequently difficult to differentiate microscopically between the M. tuberculosis complex and other mycobacteria in smearsprepared directly from specimens.Sensitivity of direct smearshas been reported to be between 22 and 43%, and smearsare therefore poor predictorsof the presenceof mycobacteria.Sensitivitymay be increased by utilization of the fluorochrome stain, concentration of specimens,and examinationof multiple

CUMITECH

16A

DIAGNOSIS

OF THE MYCOBACTERIOSES

31

specimens(6, 29, 84). Additionally, the Kinyoun stain shouldbe usedif membersof the 1M.chelonae and M fortuitum groupsare suspected,since theserapidly growingmycobacteriamay not take up the fluorochromestain.Thesespecimens, however, may stainpoorly by this method aswell. Infrequently, multiple speciesof mycobacteria may be presentin someclinical specimens.Thus, identification of a single speciesin a liquid-medium culture by direct testing with a specific geneticprobeor the differentiation of NTM by the NAP test doesnot necessarilyrule out the presenceof other speciesor of M tuberculosis, respectively. Other morphologiesmay later becomerecognizable upon subculture from liquid to solid media. Therefore, attention must be turned to ascertainingthe absenceof multiple microscopic morphologiesin primary liquid-mediumcultures.

(iii) Manual rotation of all vials before a test run diminishesthe chanceof the needlereentering the samepoint on the septum,which may have been contaminatedby a previousentry-exit. (iv) New sterile needles should be used for gassing inoculated vials. Needles should be changed,cleaned,and inspecteddaily, and those showing any damageor dullnessshould be replaced.The needletemperatureshouldreach250 to 300°C during a minimum 80-s heating cycle. The needleheater unit shouldbe cleanedmonthly and replaced periodically depending on the amount of use. (v) To detect possiblecarryover and reducethe potential for accidentalexposure,an uninoculated vial can be processedasthe final vial of eachrun. This step not only adds a final heating cycle but also allowsthe vial to be incubatedfor detection of any cross-contamination.Unfortunately, when Cross-Contamination cross-contaminationoccurs, it does so sporadiCross-contaminationof culture tubes,vials, or cally, skippingseveraltubesor vials, with up to 11 tubeswith mycobacteriacan occur in the labora- skippedvials being reported (26, 83, 107). tory, causingspuriousand potentially dangerous Significance of NTM results. Proceduresfor rapidly discovering any The significance of NTM in clinical specimens cross-contaminationthat may have occurred should be implemented.All culture tubes and may be difficult to ascertain becauseof their vials shouldbe inoculatedandplacedin numerical ubiquity in the environment and their comparaorder for testing and observation,with the order tively diminished associationwith symptomatic schedule being documented. Positive cultures disease.Criteria consideredusefulfor interpretawithin a run with previouspositive tubes or vials tion of the role of NTM in a diseaseprocess shouldbe carefully evaluatedfor the possibilityof include the following (102, 137, 138): carryover contamination. Decision making re(i) evidenceof diseaseby clinical androentgengarding the significanceof an isolate can be ographic means; helpedby direct smearobservations,positive re(ii) histopathologic evidence of presence of sult timing, and clinical correlations.It must be mycobacteria and their contribution to the patholemphasizedthat laboratory-derived data should “Yi.) . l t. alwaysbe correlated to the patient’s clinical conm isoa ion of the identical organismfrom a dition. Someworkers have expressedspecialconcern singlesite repeatedly over an extended period; (iv) increasednumbersof organismswithin the about the possibility of cross-contaminationof specimenas ascertainedby semiquantitativeculculture vials due to inadequatelysterilized sampling needlesin the BACTEC 460TB System(26, ture or direct smear; (v) isolation from or detection in a normally 83). Such contamination,although probably rare, sterile site; can be detected by the ordered testing method (vi) predilection of the speciesof mycobacteria outlined below and can be reduced by strict adisolatedto causediseaseat this site; herenceto maintenanceprotocolsoutlined by the (vii) absenceof other identifiable causesof manufacturer.The following precautionscan furdiseases; and ther minimize the chancesof contamination oc(viii) presenceof risk factors in the patient. curring. (i) Testingof vials shouldfollow an appropriate schedule,with positive vials being removed from the regular testing process.Any positive vials requiring testingshouldbe processedonly after all other vials have beentested.Needlesetsthat have beenusedon positivevials shouldbe removedfor cleaningand sterilization. (ii) To lessenthe chanceof depositionof organismson a vial’sseptum,inoculatedvials shouldnot be inverted.

Quality

Control

Considerations

Each lot number and shipment of solid and liquid media should be observed for signs of deterioration (i.e., contamination, drying, agar splitting) and evaluatedfor performancecapabilities (i.e., adequacyof mycobacterialgrowth and morphologiccharacteristics,inhibition of nonmycobacterial organismswhen appropriate). Some workers recommendM tuberculosis, MAC, M kansasii. and M fortuitum for evaluation of isola-

32

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tion media (81, 98, 110).M. haemophilum may be usedto check media containinghemin, 2% ferric ammoniumcitrate, or other appropriate factors required for its growth. P. aeruginosa, Escherichia coli, and Candida albicans may be usedasneeded to test the inhibitory capabilitiesof selectivemedia. Lots of media being evaluated should be withheld from routine use for approximately 2 weeks,within which time resultsnormally become available (98). Alternatively, manufacturers’recommendationsmaybe strictly followed whentesting mediaobtainedfrom commercialsourcesthat provide documentationof adequatequality control evaluationof the mediaprior to its shipment. Suchmediashouldstill be carefully evaluatedand tested according to the manufacturers’instructions, when available, to ensurethat damageor degradation did not occur during shipment or storageprior to arrival in the laboratory. Exposureof somemedia(suchasMiddlebrook 7HlO and 7Hll) to storage beyond 5 weeks (especiallyin direct light) may be associatedwith formation of formaldehydewithin the media. To usesuchmediabefore the possibleaccumulation of inhibitory factors, it may be incorporated into routine use approximately 1 week after inoculation with quality control organisms.However, clinical specimensbeing cultured concurrently shouldbe savedby storing remainingportions of the concentrateat 4 to 8°Cfor severalweeksuntil the resultsof quality control studiesare known (98, 110). Identification and Susceptibility Studies When studyingbiochemicaland/or characteristic growth patternson media,appropriate control organismsshouldbe inoculatedconcurrently with the unknownisolatesto obtain both positive and negativeresults.Incubation of tests and controls not only ensuresthe adequacyof the studiesbut also allows visual comparison of positive and negative reactions with those produced by the organismbeing identified. Individual componentsof basalmedia for susceptibility studies vary substantially and affect studyresults(46). New and old lots of mediamay be comparedby a grading systemthat evaluates not only numbersbut alsosizesof coloniesinoculated to both systems.Susceptibleand resistant control organismsshouldbe testedwhen susceptibility studiesare done.It hasbeenrecommended that a strain of M. tuberculosis susceptibleto all antimycobacterialagentsand a strain of M. kansasii susceptibleto high concentrationsbut resistant to low concentrationsof isoniazidbe included for eachlot of test media (98, 110). Manufacturers’ recommendationsshould be followed when commercialsystemssuch as the BACTEC are usedfor susceptibilitystudies.

CUMITIXH

16~4

Equipment Documentation of a monitoring system for equipment should be present in the laboratory. The systemshouldensurethat equipmentis functioning with appropriateaccuracyand precisionas delineated by the manufacturer’s specifications. Biological safety cabinetsshouldbe certified by a qualified certifying agency at least annually. To ascertain proper airflow on a daily basis, thin stripsof single-layertissuepaper can be taped to eachsideof the front of a cabinet.The sametype of strips can be used to qualitatively ascertain airflow into negative-pressureisolation rooms. If UV lightsare present,they shouldbe dusteddaily and wiped cleanwith alcohol every 2 weeks(58). Brushesand bearingsin centrifugesshouldbe checked at least biannually, and the revolutions per minute shouldbe monitored with a tachometer. The relative centrifugal force exerted can directly influencerecovery rates of mycobacteria, with centrifugal forces over 3,000 X g and approaching 3,800 X g being necessaryfor optimal yield (110). Extending centrifugation time at lower centrifugal force causesheating of the system, which mayprove deleteriousto the mycobacteria. Thus, many laboratories use refrigerated centrifugeswhenprocessingspecimensat higherg forces. All incubators,refrigerators,freezers,andwater baths shouldbe monitored daily for temperature constancy and cleaned regularly every 1 to 6 months(dependingon the equipment)(58). Incubatorswith CO2 content shouldbe checkeddaily with a systemsuch as the Fyrite CO2 analyzer. Light shouldbe controlled in the incubator. Thermometersare best placedin a water reservoir for temperature recording and should be checkedannually againsta thermometercertified by the National Bureau of Standards.Equipment such as microscopesand pH meters should be kept clean and serviced regularly to maintain appropriate performance. The fluorescent light source in the fluorescent microscopeshould be monitored, serviced,and replacedassuggested by the manufacturerof the system. Nebulizersusedfor sputuminduction shouldbe rinsed with distilled water and soakedin a 2% acetic acid solution for at least 10 min. Finally, they can be rinsedwith water and dried in air. The effluent can be cultured as a test for sterility (6). CONCLUSION Diagnostic strides in the detection, identification, and susceptibilitytesting of M. tuberculosis and NTM continue to be made. The antibiotic gradient strip, E test, hasbeen adapted for susceptibility testing of mycobacteria(127), and the Mycobacteria Growth IndicatorTubessystem(Becton DickinsonMicrobiology Systems)(47), strand

CUMITECH

16A

DIAGNOSIS

displacement amplification (Becton DickinsonMicrobiology Systems)(go), and a new Gen-Probe amplified Mycobacterium direct test (23) show promisefor early detection and identification of M. tuberculosis in the future.

19. 20.

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